Method of treating microbial infections

ABSTRACT

The disclosure provides for novel antimicrobial agents, methods of making, and methods of use thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/750,979, filed Jun. 25, 2015, which is a divisional of U.S. patentapplication Ser. No. 13/985,548, filed Aug. 14, 2013, which is a U.S.National Stage application filed under 35 U.S.C. §371 and claimspriority to International Application No. PCT/US2012/025044, filed Feb.14, 2012, which application claims priority under 35 U.S.C. §119 fromProvisional Application Ser. No. 61/443,149, filed Feb. 15, 2011 andfrom Provisional Application Ser. No. 61/482,590, filed May 4, 2011, thedisclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant No.AI083358, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

TECHNICAL FIELD

The disclosure relates to anti-microbial agents, methods of making, andmethods of use thereof.

BACKGROUND

Ogston (1881) coined the genus Staphylococcus to describe grapelikeclusters of bacteria (staphylo=grape, Gr.) recovered in pus fromsurgical abscesses. Entering its seventh decade, the era ofantimicrobial therapy has greatly reduced morbidity and mortality frominfectious diseases. However, the emergence of resistant microorganismshas now reached epidemic proportions and poses great challenges to themedical community. Worrisome trends are particularly evident in thepre-eminent Gram-positive bacterial pathogen S. aureus, which has becomeincreasingly unresponsive to first-line antibiotic therapies. S. aureusis probably the most common cause of life-threatening acute bacterialinfections in the world, and is capable of causing a diverse array ofdiseases, ranging in severity from a simple boil or impetigo tofulminant sepsis or toxic shock syndrome. S. aureus is the singleleading cause of bacteremia, hospital-related (nosocomial) infections,skin and soft tissue infections, wound infections, and bone and jointinfections. It is one of the most common agents of endocarditis and foodpoisoning.

National prospective surveillance of over 24,000 invasive bacterialisolates show disease-associated S. aureus strains with methicillinresistance (MRSA) have increased from 22% in 1995 to 57% currently. MRSAare now frequently identified in community-acquired infections as wellas in hospital settings. A half-century of synthesizing analogs based on<10 antibacterial scaffolds has resulted in the development andmarketing of >100 antibacterial agents but, with the exception of theoxazolidinone core, no new scaffolds have emerged in the past 30 yearsto address the emerging resistance problems.

Classic antibiotic approaches attempt to kill or suppress growth ofbacteria by targeting essential cell functions such as cell wallbiosynthesis, protein synthesis, DNA replication, RNA polymerase, ormetabolic pathways. These conventional therapies run a high risk oftoxicity since many of these cell functions are also essential tomammalian cells and require fine molecular distinction between themicrobial target and the host cell counterpart(s). Second, therepetitive use of the same targets means that when a bacterium evolvesresistance to a particular antibiotic agent during therapy, it canbecome simultaneously cross resistant to other agents acting on the sametarget, even though the bacterium has never been exposed to the otheragents. Third, conventional therapies exert a “life-or-death” challengeupon the bacterium, and thus a strong selective pressure to evolveresistance to the antimicrobial agent. Finally, many current antibioticshave very broad spectrums of activity, with the side effect oferadicating many components of the normal flora, leading to undesiredcomplications such as Clostridium difficile colitis or secondary fungalinfections (e.g. Candida).

The emergence of MRSA has compromised the clinical utility ofmethicillin and related antibiotics (oxacillin, dicloxacillin) and allcephalosporings (e.g. cefazolin, cephalexin) in empiric therapy of S.aureus infections. MRSA often have significant levels of resistance tomacrolides (e.g. erythromycin), beta-lactamase inhibitor combinations(e.g. Unasyn, Augmentin) and fluoroquinolones (e.g. ciprofloxacin), andare occasionally resistant to clindamycin, trimethoprim/sulfamethoxisol(Bactrim), and rifampin. In serious S. aureus infection, intravenousvancomycin is the last resort, but there have now also been alarmingreports of S. aureus resistance to vancomycin, an intravenous antibioticcommonly used to treat MRSA.

New anti-MRSA agents such as linezolid (Zyvox® orquinupristin/dalfopristin (Synercid®), both of which utilize thetraditional target of binding to the ribosomal subunits to inhibit RNAsynthesis are prohibitively expensive.

Existing antibiotic therapies non-specifically kill the majority ofskin-residing bacteria, disrupting the homeostasis of skin residentmicroflora. For example, benzoyl peroxide (BPO) is one of the mostfrequently used topical medications. BPO strongly suppresses the growthof S. epidermidis. S. epidermidis contributes to the skin residentmicroflora-based defense of the skin epithelium. The imbalance ofmicroflora could contribute to the pathogenesis of skin inflammatorydiseases, such as atopic dermatitis, rosacea and acne vulgaris etc.

SUMMARY

The disclosure also provides a method of preparing and substantiallypurifying a “firmocidin”, a compound of the disclosure. In oneembodiment, the method comprises extracting a flow-through from a 3Kcut-off of the media and performing organic extraction, followed byseparating on an NH₂-100 column eluted with a linear gradient of waterin acetonitrile for 25 min. In a more specific embodiment, the methodcomprises obtaining S. epidermidis culture media, collecting theflow-through of the media using a 3K cut-off membrane, lyophilizing thecollected flow through, resuspending the lyophilized product in methanoland partitioning in 90% acetonitrile, collecting the organic phase,lyophilizing the organic phase, resuspending the lyophilized organicphase in an aqueous media, filtering on a C₁₈ sep-pak cartridge,lyophilizing and resuspending in methanol, collecting the supernatant,lyophilizing, partitioning in acetonitrile and collecting thesupernatant, and separating on Taskgel NH₂-100 column eluted with alinear gradient of 5-20% water in acetonitrile for 25 min. Thedisclosure also provides a composition obtained by the foregoing method,wherein the composition has anti-microbial activity.

In a particular embodiment, the disclosure provides for a firmocidincompound selected from the group comprising:

(a) Formula I:

wherein:

X¹-X¹⁰ are each independently either a C, N or O;

R¹-R¹⁸ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence;

(b) Formula II

wherein:

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹-R³⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence;

(c) Formula III

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵-R⁵⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence;

(d) Formula IV

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵-R⁶⁹ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence;

derivatives or analogs of Formulas I-IV thereof, includingpharmaceutical salts and prodrugs; and

wherein the compound has antimicrobial activity.

In another embodiment, the disclosure provides for compound comprisingat least 5 carbon atoms, at least 5 nitrogen atoms, at least 5 hydrogenatoms, and at least one oxygen atom and is selected from the groupcomprising:

(a) Formula I:

wherein:

X¹-X¹⁰ are each independently either a C, N or O;

R¹-R¹⁸ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence;

(b) Formula II

wherein:

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹-R³⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence;

(c) Formula III

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵-R⁵⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence;

(d) Formula IV

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵-R⁶⁹ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence;

derivatives or analogs of Formulas I-IV thereof, includingpharmaceutical salts and prodrugs; and

wherein the compound has antimicrobial activity.

In a further embodiment, the disclosure provides for compound comprisingat least 5 carbon atoms, at least 5 nitrogen atoms, at least 5 hydrogenatoms, and at least one oxygen atom and is selected from the groupcomprising:

(a) Formula I(a):

wherein:

X¹-X¹⁰ are each independently either a C, N or O;

R¹, R³, R⁷, R⁹, R¹¹, R¹³, R¹⁵, and R¹⁷ are each independently selectedfrom the group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence;

(b) Formula II(a)

wherein:

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹, R²³, R²⁵, R²⁷, R²⁹, R³¹, and R³³ are each independently selectedfrom the group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence;

(c) Formula III(a)

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵, R³⁷, R⁴¹, R⁴³, R⁴⁵, R⁴⁷, R⁴⁹, R⁵¹, and R⁵³, are each independentlyselected from the group comprising H, D, optionally substituted(C₁-C₂)alkyl, optionally substituted (C₁-C₂)alkenyl, optionallysubstituted (C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence;

(d) Formula IV(a)

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵, R⁵⁷, R⁵⁹, R⁶¹, R⁶³, R⁶⁵, R⁶⁷, and R⁶⁹ are each independentlyselected from the group comprising H, D, optionally substituted(C₁-C₂)alkyl, optionally substituted (C₁-C₂)alkenyl, optionallysubstituted (C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence;

derivatives or analogs of Formulas I(a)-IV(a) thereof, includingpharmaceutical salts and prodrugs; and

wherein the compound has antimicrobial activity.

In yet a further embodiment, the disclosure provides for a compoundselected from the group comprising:

In a certain embodiment, the disclosure provides for a compound selectedfrom the group comprising:

In another embodiment, the disclosure provides for a compound having astructural formula of:

In yet another embodiment, the disclosure provides for a compound havinga structural formula of:

The disclosure also provides a method to use any one or more of thecompounds above to treat MRSA, as well as group A streptococcus (GAS)and group B streptococcus (GBS) and Staphylococcus aureus (S. aureus)infections, which are the most common pathogens in human skin. Thus, afirmocidin of the disclosure can be used for antibiotic treatment tocontrol skin infections.

In a particular embodiment, the disclosure provides for inhibiting thegrowth of a bacterium or fungus comprising contacting the bacterium orfungus with an inhibiting effective amount of a composition comprising acompound disclosed herein. In another embodiment, the disclosureprovides for contacting the bacterium or fungus is either in vitro or invivo, such as where the contacting in vivo is through topicaladministration. In a further embodiment, the disclosure provides forinhibiting the growth of a bacterium that is either gram positive orgram negative. In another embodiment, the disclosure provides for acomposition comprising the compound disclosed herein with at least oneadditional antimicrobial agent. In yet another embodiment, thedisclosure provides for a composition comprising the compound disclosedherein with a pharmaceutically acceptable carrier.

As described more fully herein, firmocidin suppresses growth ofmethicillin-resistant S. aureus (MRSA), a strain that is highlyresistant to some antibiotics. Existing antibiotic therapies cannon-specifically kill bacteria, which may disrupt the homeostasis ofskin-resident microflora. However, Firmocidin does not affect the growthof S. epidermidis which contributes normal defense at the skinepithelium. Firmocidin also did not affect viability of humankeratinocytes and sebocytes. In addition, Firmocidin is isolated from amicroorganism residing in the normal skin microflora, suggesting lowtoxicity to the host. Thus, Firmocidin can be safely used as apathogen-specific antibiotic therapy for skin infections.

In a particular embodiment, the disclosure provides a method fortreating an infection, including infections caused by bacteria, fungus,parasites or viruses, or a dermatological disorder, includingdermatological disorders such as, wounds, diabetic ulcers, acne,rosacea, atopic dermatitis, pyodermas, and burn wounds, by administeringan effective amount of a firmocidin compound disclosed herein. Thedisclosure also provides for a composition formulated for systemic ortopical administration.

In another embodiment, the Firmocidin of the disclosure comprises an NMRspectra as set forth in FIGS. 9 and 10. The firmocidin NMR did not matchany previously characterized antimicrobial agents, suggesting firmocidinis a novel antimicrobial component. In vitro bactericidal assaysrevealed that firmocidin showed antimicrobial activity against Group Astreptococcus, Group B streptococcus and Staphylococcus aureus, whichare the most common pathogens in the human skin.

The disclosure provides for a firmocidin compound that has approximatelythe physical and structural properties as presented in FIGS. 1-10 andthe biological properties as presented in FIGS. 12-14.

In a certain embodiment, a pharmaceutical composition comprising afirmocidin compound disclosed herein further comprises one or morepharmaceutically acceptable carriers. In another embodiment, thedisclosure provides for a pharmaceutical composition comprising acompound disclosed herein which is formulated for oral, parenteral, ortopical administration. In another embodiment, the disclosure providesfor a pharmaceutical composition comprising a compound disclosed hereinfor topical administration wherein the topical dosage form is either inthe form of a cream, ointment, gel, spray or lotion.

In a certain embodiment, the disclosure provides for a compositioncomprising a compound disclosed herein and further comprises one or moreadditional therapeutic agents, including antibiotics, sepsis treatments,steroidal drugs, anti-fungal agents, and antipruritics. Examples ofantibiotics, include, but are not limited to, amoxicillin, ampicillin,arsphenamine, azithromycin, aztreonam, azlocillin, bacitracin,carbenicillin, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin,cefdinir, cefditorin, cefepime, cefixime, cefoperazone, cefotaxime,cefoxitin, cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, chloramphenicol, cilastin, ciprofloxacin,clarithromycin, clindamycin, clofazimine, cloxacillin, colistin,dalfopristan, demeclocycline, dicloxacillin, dirithromycin, doxycycline,erythromycin, enafloxacin, enviomycin, ertepenem, ethambutol,flucloxacillin, fosfomycin, furazolidone, gatifloxacin, geldanamycin,gentamicin, herbimicin, imipenem, linezolid, lomefloxacin, loracarbef,mafenide, moxifloxacin, meropenem, metronidazole, mezlocillin,minocycline, mupirozin, nafcillin, neomycin, netilmicin, nitrofurantoin,norfloxacin, oxytetracycline, penicillin, piperacillin, platensimycin,polymixin B, prochlorperazine, prontocil, quinupristine, rifabutin,roxithromycin, spectinomycin, sulfacetamide, sulfamethizole,sulfamethoxazole, teicoplanin, telithromycin, tetracycline,thioacetazone, thioridazine, ticarcillin, tobramycin, trimethoprim,troleandomycin, trovafloxacin, and vancomycin.

In yet another embodiment, the disclosure provides a method for thetreatment, prevention, or amelioration of one or more symptoms of aninfection by a foreign agent or a dermatological disorder comprisingadministering a therapeutically effective amount of a firmocidincompound disclosed herein. Examples of foreign agents, include, but arenot limited to, bacterium, parasite, virus, or fungus. Bacteria that canbe affected by the use of a compound, derivative or analog thereof,including pharmaceutical salt and prodrug forms, include bothgram-negative and gram-positive bacteria. For example, bacteria that canbe affected include Staphylococcus aureus, Streptococcus pyogenes (groupA), Streptococcus sp. (viridans group), Streptococcus agalactiae (groupB), S. bovis, Streptococcus (anaerobic species), Streptococcuspneumoniae, and Enterococcus sp.; Gram-negative cocci such as, forexample, Neisseria gonorrhoeae, Neisseria meningitidis, and Branhamellacatarrhalis; Gram-positive bacilli such as Bacillus anthracis, Bacillussubtilis, P. acne Corynebacterium diphtheriae and Corynebacteriumspecies which are diptheroids (aerobic and anerobic), Listeriamonocytogenes, Clostridium tetani, Clostridium difficile, Escherichiacoli, Enterobacter species, Proteus mirablis and other sp., Pseudomonasaeruginosa, Klebsiella pneumoniae, Salmonella, Shigella, Serratia, andCampylobacter jejuni. In a preferred embodiment, the bacterium isselected from the group comprising Group A streptococcus (GAS), Group Bstreptococcus (GBS), and S. aureus.

In another embodiment, the disclosure provides a method of treatment foran infection caused by a fungus by administering a compound describedherein, derivative or analog thereof, including pharmaceutical salt andprodrug forms. Examples of fungal organisms may be affected, include forexample, Microsporum canis and other Microsporum sp.; Trichophyton sp.such as T. rubrum, and T. mentagrophytes, yeasts, e.g., Candidaalbicans, C. Tropicalis, or other Candida species, Saccharomycescerevisiae, Torulopsis glabrata, Epidermophyton floccosum, Malasseziafurfur (Pityropsporon orbiculare, or P. ovale, Cryptococcus neoformans,Aspergillus fumigatus, Aspergillus nidulans, and other Aspergillus sp.,Zygomycetes, e.g., Rhizopus, Mucor, Paracoccidioides brasiliensis,Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis,and Sporothrix schenckii.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a flow chart for the preparation of firmocidin from aculture supernatant of S. epidermidis.

FIG. 2A-B presents the purification of firmocidin from the culturesupernatant of S. epidermidis by HPLC and the activity of certainindicated fractions. The crude culture supernatant S. epidermidis wasapplied to a TaskGel NH₂-100 column (4.5 mm×150 mm) and eluted with a25-min linear gradient of 5-20% H₂O in acetonitrile at a flow rate of 1ml/min; the eluent was monitored at 270 nm (FIG. 2A). Fractions 26-31were lyophilized, reconstituted in PBS and applied on an agar plateinoculated with GAS (NZ131) (FIG. 2B). Clear zones demonstrateantimicrobial activity.

FIG. 3 presents a chromatogram of firmocidin using high-resolutionESI-FT-MS. The mass spectra indicate that firmocidin has a molecularmass of 152.0567. Based on the mass spectrum, firmocidin has a predictedstructure of 5 carbon atoms, 5 hydrogen atoms, 5 nitrogen atoms, and 1oxygen atom.

FIG. 4A-B presents (A) a high-resolution ESI-FT-MS spectrum verifyingthat firmocidin has a predicted molecular mass of 152.0567; and (B) ESIpositive ion mode MS/MS analysis on the m/z peak of 152.0567.

FIG. 5 presents a side by side comparison of the high-resolutionHR-ESI-FT-MS spectra for firmocidin and guanine. The difference andsimilarities in the major electron impact fragmentation of firmocidinversus guanine is readily apparent.

FIG. 6 presents EI-MS spectra showing the difference between firmocidinand guanine. The mass spectra show that firmocidin has fragment peaksthat are present and not present in the guanine chromatogram.

FIG. 7 presents mass spectrum and major electron impact fragmentation ofguanine. Wherein, fragment peaks at 69.2 and 110.1 of the guanine massspectrum are not present in the mass spectrum for firmocidin.

FIG. 8 presents structural models demonstrating select electron impactfragments that are common to both fimocidin and guanine and electronimpact fragments that are guanine specific. Atoms encompassed in thedashed ovals represent the major fragment observed along with thepredicted mass of the fragment. The guanine specific fragment m/z peakat 110.1 is a singular fragment containing the atoms resulting from thecombination of the two dashed ovals.

FIG. 9 presents a ¹H NMR spectrum of firmocidin. The chemical shiftsobtained are identical to an adenine-N-oxide.

FIG. 10 presents a ¹H NMR spectrum of firmocidin. The characteristicnuclear shifts of a benzene ring appear at 8.0-8.5 ppm.

FIG. 11 provides that compounds labeled 1-7, which have the molecularformula of C₅H₅N₅O, but were not antimicrobial agents for GAS. Eachcompound was dissolved or suspended in PBS at 2 mg/mL and 5 mL wasapplied on an agar plate inoculated with GAS (NZ131).

FIG. 12 presents data demonstrating firmocidin inhibits growth ofvarious pathogens but not S. epidermidis. The data also show thatfirmocidin antimicrobial activity is superior to benzoyl peroxide (BPO).HPLC-purified firmocidin was incubated with various bacteria (1×10⁶CFU/mL) in the indicated medium for 7 hrs. BPO was used as a positivecontrol. After incubation, OD₆₀₀ was determined to estimate bacteriagrowth.

FIG. 13 presents data showing firmocidin's bactericidal activity againstGAS and bacteriostatic activity against GBS and S. aureus, but not S.epidermidis. Various bacteria (1×10⁶ CFU/mL) were incubated withHPLC-purified firmocidin (0, 20 and 60 mg/mL) in the indicated medium.Colony-forming unit (CFU) of the bacteria was determined at theindicated times by plating serial dilutions of bacteria suspension.

FIG. 14 presents data demonstrating that firmocidin does not have adeleterious effect on the cell viability of human keratinocytes andsebocytes. The immortalized human HaCaT keratinocytes and SZ95 sebocytes(1×10⁵ cells/well) were incubated with indicated concentrations offirmocidin for 24 hr at 37° C. As a background, Triton X-100 (0.1%(v/v)) was added to achieve 0% cell viability. After incubation, cellviabilities were determined with a MTT assay.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes a plurality of such compounds and reference to “the cell”includes reference to one or more cells and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods andreagents similar or equivalent to those described herein can be used inthe practice of the disclosed methods and compositions, the exemplarymethods and materials are now described.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,”“including,” “have,” “haves,” and “having” are interchangeable and notintended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

All publications mentioned herein are incorporated herein by referencein full for the purpose of describing and disclosing the methodologies,which are described in the publications, which might be used inconnection with the description herein. However, with respect to anysimilar or identical terms found in both the incorporated publicationsor references and those expressly put forth or defined in thisapplication, then those terms definitions or meanings expressly putforth in this application shall control in all respects. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The term “alkyl”, refers to an organic group that is comprised of carbonand hydrogen atoms that contains single covalent bonds between thecarbons. Generally, an “alkyl” as used in this disclosure, refers to anorganic group that contains 1 to 20 carbon atoms, unless statedotherwise. Wherein if there is more than 1 carbon, the carbons may beconnected in a linear manner, or alternatively if there are more than 2carbons then the carbons may also be linked in a branched fashion sothat the parent chain contains one or more secondary, tertiary, orquaternary carbons. An alkyl may be substituted or unsubstituted, unlessstated otherwise. Substituted alkyl groups include among others thosewhich are substituted with aryl groups, which in turn can be optionallysubstituted. Specific alkyl groups include methyl, ethyl, n-propyl,iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl,n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, andcyclohexyl groups, all of which are optionally substituted. Specificsubstituted alkyl groups include haloalkyl groups, particularlytrihalomethyl groups and specifically trifluoromethyl groups.

The term “alkenyl”, refers to an organic group that is comprised ofcarbon and hydrogen atoms that contains at least one double covalentbond between two carbons. Generally, an “alkenyl” as used in thisdisclosure, refers to organic group that contains 1 to 20 carbon atoms,unless stated otherwise. While a C₁-alkenyl can form a double bond to anatom of a parent chain, an alkenyl group of three or more carbons cancontain more than one double bond. It certain instances the alkenylgroup will be conjugated, in other cases an alkenyl group will not beconjugated, and yet other cases the alkenyl group may have stretches ofconjugation and stretches of nonconjugation. Additionally, if there ismore than 1 carbon, the carbons may be connected in a linear manner, oralternatively if there are more than 3 carbons then the carbons may alsobe linked in a branched fashion so that the parent chain contains one ormore secondary, tertiary, or quaternary carbons. An alkenyl may besubstituted or unsubstituted, unless stated otherwise. Substitutedalkenyl groups include among others those which are substituted withalkyl or aryl groups, which groups in turn can be optionallysubstituted. Specific alkenyl groups include ethenyl, prop-1-enyl,prop-2-enyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, branchedpentenyl, hex-1-enyl, branched hexenyl, all of which are optionallysubstituted.

The term “alkynyl”, refers to an organic group that is comprised ofcarbon and hydrogen atoms that contains a triple covalent bond betweentwo carbons. Generally, an “alkynyl” as used in this disclosure, refersto organic group that contains 1 to 20 carbon atoms, unless statedotherwise. While a C₁-alkynyl can form a triple bond to an atom of aparent chain, an alkynyl group of three or more carbons can contain morethan one triple bond. Where if there is more than 1 carbon, the carbonsmay be connected in a linear manner, or alternatively if there are morethan 4 carbons then the carbons may also be linked in a branched fashionso that the parent chain contains one or more secondary, tertiary, orquaternary carbons. An alkynyl may be substituted or unsubstituted,unless stated otherwise.

The term “cycloalkyl”, as used in this disclosure, refers to an alkylthat contains at least 3 carbon atoms but no more than 12 carbon atomsconnected so that it forms a ring. A “cycloalkyl” for the purposes ofthis disclosure encompass from 1 to 7 cycloalkyl rings, wherein when thecycloalkyl is greater than 1 ring, then the cycloalkyl rings are joinedso that they are linked, fused, or a combination thereof. A “cycloalkyl”can also include bicyclic and tricyclic-based groups. A cycloalkyl maybe substituted or unsubstituted, or in the case of more than onecycloalkyl ring, one or more rings may be unsubstituted, one or morerings may be substituted, or a combination thereof.

The term “cycloalkenyl”, as used in this disclosure, refers to an alkenethat contains at least 3 carbon atoms but no more than 12 carbon atomsconnected so that it forms a ring. A “cycloalkenyl” for the purposes ofthis disclosure encompass from 1 to 7 cycloalkenyl rings, wherein whenthe cycloalkenyl is greater than 1 ring, then the cycloalkenyl rings arejoined so that they are linked, fused, or a combination thereof“Cycloalkenyl” can include bicyclic and tricyclic-based groups. Acycloalkenyl may be substituted or unsubstituted, or in the case of morethan one cycloalkenyl ring, one or more rings may be unsubstituted, oneor more rings may be substituted, or a combination thereof. Specificalkenyl groups include cycloprop-1-enyl, cyclobut-1-enyl,cyclobut-2-enyl, cyclopent-1-enyl, cyclohexenyl, all of which areoptionally substituted.

The term “aryl”, as used in this disclosure, refers to a conjugatedplanar ring system with delocalized pi electron clouds that contain onlycarbon as ring atoms. An “aryl” for the purposes of this disclosureencompass from 1 to 7 aryl rings wherein when the aryl is greater than 1ring the aryl rings are joined so that they are linked, fused, or acombination thereof. An aryl may be substituted or unsubstituted, or inthe case of more than one aryl ring, one or more rings may beunsubstituted, one or more rings may be substituted, or a combinationthereof. Substituted aryl groups include among others those which aresubstituted with alkyl or alkenyl groups, which groups in turn can beoptionally substituted. Specific substituted aryl groups include mono-,di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di-,tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalenegroups; 3- or 4-halo-substituted phenyl groups, 3- or4-alkyl-substituted phenyl groups, 3- or 4-alkoxy-substituted phenylgroups, 3- or 4-RCO-substituted phenyl, 5- or 6-halo-substitutednaphthalene groups. More specifically, substituted aryl groups includeacetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenylgroups, particularly 3-fluorophenyl and 4-fluorophenyl groups;chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenylgroups; methylphenyl groups, particularly 4-methylphenyl groups, andmethoxyphenyl groups, particularly 4-methoxyphenyl groups. Specific arylgroups include phenyl groups, biphenyl groups, and naphthyl groups, allof which are optionally substituted.

The term “heterocycle”, as used in this disclosure, refers to ringstructures that contain at least 1 noncarbon ring atom. A “heterocycle”for the purposes of this disclosure encompass from 1 to 7 heterocyclerings wherein when the heterocycle is greater than 1 ring theheterocycle rings are joined so that they are linked, fused, or acombination thereof. A heterocycle may be a hetero-aryl or nonaromatic,or in the case of more than one heterocycle ring, one or more rings maybe nonaromatic, one or more rings may be hetero-aryls, or a combinationthereof. A heterocycle may be substituted or unsubstituted, or in thecase of more than one heterocycle ring one or more rings may beunsubstituted, one or more rings may be substituted, or a combinationthereof. Typically, the noncarbon ring atom is N, O, S, Si, Al, B, or P.In case where there is more than one noncarbon ring atom, thesenoncarbon ring atoms can either be the same element, or combination ofdifferent elements, such as N and O. Examples of heterocycles include,but are not limited to: a monocyclic heterocycle such as, aziridine,oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline,imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydro-pyridine, piperazine, morpholine,thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran,1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine,2,3,4,7-tetrahydro-1H-azepine homopiperazine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethylene oxide; and polycyclicheterocycles such as, indole, indoline, isoindoline, quinoline,tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline,1,4-benzodioxan, coumarin, dihydrocoumarin, benzofuran,2,3-dihydrobenzofuran, isobenzofuran, chromene, chroman, isochroman,xanthene, phenoxathiin, thianthrene, indolizine, isoindole, indazole,purine, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, phenanthridine, perimidine, phenanthroline, phenazine,phenothiazine, phenoxazine, 1,2-benzisoxazole, benzothiophene,benzoxazole, benzthiazole, benzimidazole, benztriazole, thioxanthine,carbazole, carboline, acridine, pyrolizidine, and quinolizidine. Inaddition to the polycyclic heterocycles described above, heterocycleincludes polycyclic heterocycles wherein the ring fusion between two ormore rings includes more than one bond common to both rings and morethan two atoms common to both rings. Examples of such bridgedheterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and7-oxabicyclo[2.2.1]heptane.

The terms “heterocyclic group”, “heterocyclic moiety”, “heterocyclic”,or “heterocyclo” used alone or as a suffix or prefix, refers to aheterocycle that has had one or more hydrogens removed therefrom.

The term “heterocyclyl” used alone or as a suffix or prefix, refers amonovalent radical derived from a heterocycle by removing a hydrogentherefrom. Heterocyclyl includes, for example, monocyclic heterocyclyls,such as, aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl,thietanyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, pyrazolidinyl,pyrazolinyl, dioxolanyl, sulfolanyl, 2,3-dihydrofuranyl,2,5-dihydrofuranyl, tetrahydrofuranyl, thiophanyl, piperidinyl,1,2,3,6-tetrahydro-pyridinyl, piperazinyl, morpholinyl, thiomorpholinyl,pyranyl, thiopyranyl, 2,3-dihydropyranyl, tetrahydropyranyl,1,4-dihydropyridinyl, 1,4-dioxanyl, 1,3-dioxanyl, dioxanyl,homopiperidinyl, 2,3,4,7-tetrahydro-1H-azepinyl, homopiperazinyl,1,3-dioxepanyl, 4,7-dihydro-1,3-dioxepinyl, and hexamethylene oxidyl. Inaddition, heterocyclyl includes aromatic heterocyclyls or heteroaryl,for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl,furyl, furazanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl,isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and 1,3,4 oxadiazolyl. Additionally, heterocyclylencompasses polycyclic heterocyclyls (including both aromatic ornon-aromatic), for example, indolyl, indolinyl, isoindolinyl,quinolinyl, tetrahydroquinolinyl, isoquinolinyl,tetrahydroisoquinolinyl, 1,4-benzodioxanyl, coumarinyl,dihydrocoumarinyl, benzofuranyl, 2,3-dihydrobenzofuranyl,isobenzofuranyl, chromenyl, chromanyl, isochromanyl, xanthenyl,phenoxathiinyl, thianthrenyl, indolizinyl, isoindolyl, indazolyl,purinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, pteridinyl, phenanthridinyl, perimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxazinyl, 1,2-benzisoxazolyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benzimidazolyl,benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl,pyrolizidinyl, and quinolizidinyl. In addition to the polycyclicheterocyclyls described above, heterocyclyl includes polycyclicheterocyclyls wherein the ring fusion between two or more rings includesmore than one bond common to both rings and more than two atoms commonto both rings. Examples of such bridged heterocycles include, but arenot limited to, quinuclidinyl, diazabicyclo[2.2.1]heptyl; and7-oxabicyclo[2.2.1]heptyl.

The term “hetero-aryl” used alone or as a suffix or prefix, refers to aheterocycle or heterocyclyl having aromatic character. Examples ofheteroaryls include, but are not limited to, pyridine, pyrazine,pyrimidine, pyridazine, thiophene, furan, furazan, pyrrole, imidazole,thiazole, oxazole, pyrazole, isothiazole, isoxazole, 1,2,3-triazole,tetrazole, 1,2,3-thiadiazole, 1,2,3-oxadiazole, 1,2,4-triazole,1,2,4-thiadiazole, 1,2,4-oxadiazole, 1,3,4-triazole, 1,3,4-thiadiazole,and 1,3,4-oxadiazole.

The term “hetero-” when used as a prefix, such as, hetero-alkyl,hetero-alkenyl, hetero-alkynyl, or hetero-hydrocarbon, for the purposeof this disclosure refers to the specified hydrocarbon having one ormore carbon atoms replaced by non-carbon atoms as part of the parentchain. Examples of such non-carbon atoms include, but are not limitedto, N, O, S, Si, Al, B, and P. If there is more than one non-carbon atomin the hetero-based parent chain then this atom may be the same elementor may be a combination of different elements, such as N and O.

The term “mixed ring system” refers to optionally substituted ringstructures that contain at least two rings, and wherein the rings arejoined together by linking, fusing, or a combination thereof. A mixedring system comprises a combination of different ring types, includingcycloalkyl, cycloalkenyl, aryl, and heterocycle.

The term “unsubstituted” with respect to hydrocarbons, heterocycles, andthe like, refers to structures wherein the parent chain contains nosubstituents.

The term “substituted” with respect to hydrocarbons, heterocycles, andthe like, refers to structures wherein the parent chain contains one ormore substituents. For example, optionally substituted hydrocarbons,hetero-hydrocarbons, heterocycles, mixed ring systems, and the like, caninclude substitution with one or more of the following substituents:halogens, CN, —COOR, —OR, —COR, —OCOOR, —CON(R)₂, —OCON(R)₂, —N(R)₂,NO₂, —SR, —SO₂R, —SO₂N(R)₂ or —SOR groups, wherein R is selected fromthe group comprising a hydrocarbon, a hetero-hydrocarbon, heterocycle,and mixed ring system. Optional substitution of alkyl groups includessubstitution with one or more alkenyl groups, aryl groups or both,wherein the alkenyl groups or aryl groups are also optionallysubstituted. Optional substitution of alkenyl groups includessubstitution with one or more alkyl groups, aryl groups, or both,wherein the alkyl groups or aryl groups are also optionally substituted.Optional substitution of aryl groups includes substitution of the arylring with one or more alkyl groups, alkenyl groups, or both, wherein thealkyl groups or alkenyl groups are also optionally substituted.

The term “substituent” refers to an atom or group of atoms substitutedin place of a hydrogen atom. For purposes of this disclosure, asubstituent would include deuterium atoms.

Optional substituents for hydrocarbons, hetero-hydrocarbons,heterocycles, mixed ring systems, and the like, include among others:

—COOR where R is a hydrogen or an alkyl group or an aryl group and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which are optionally substituted;

—COR where R is a hydrogen, or an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, or phenyl groupsall of which groups are optionally substituted;

—CON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—OCON(R)₂ where each R, independently of each other R, is a hydrogen oran alkyl group or an aryl group and more specifically where R is methyl,ethyl, propyl, butyl, or phenyl groups all of which groups areoptionally substituted; R and R can form a ring which may contain one ormore double bonds;

—N(R)₂ where each R, independently of each other R, is a hydrogen, or analkyl group, acyl group or an aryl group and more specifically where Ris methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of whichare optionally substituted; or R and R can form a ring which may containone or more double bonds;

—SR, —SO₂R, or —SOR where R is an alkyl group or an aryl groups and morespecifically where R is methyl, ethyl, propyl, butyl, phenyl groups allof which are optionally substituted; for example —SR, R can be hydrogen;

—OCOOR where R is an alkyl group or an aryl groups;

—SO₂N(R)₂ where R is a hydrogen, an alkyl group, or an aryl group and Rand R can form a ring; and

—OR where R═H, alkyl, aryl, or acyl; for example, R can be an acylyielding —OCOR* where R* is a hydrogen or an alkyl group or an arylgroup and more specifically where R* is methyl, ethyl, propyl, butyl, orphenyl groups all of which groups are optionally substituted.

As used herein, a wavy line intersecting another line that is connectedto an atom indicates that this atom is covalently bonded to anotherentity that is present but not being depicted in the structure. A wavyline that does not intersect a line but is connected to an atomindicates that this atom is interacting with another atom by a bond orsome other type of identifiable association.

A bond indicated by a straight line and a dashed line indicates that thebond may be a single covalent bond or alternatively a double covalentbond. But in the case where a ring atom's maximum valence would beexceeded by forming a double covalent bond with another ring atom, thenthe bond would be a single covalent bond.

For the purposes of this disclosure, in the instance that a ring atomdesignated as X would exceed its maximum valence by binding a groupdesignated by R, then the group designated by R would be absent.

The term “antimicrobial” as it relates to treatments, agents, andcompounds refers to an agent that can be used to suppress, attenuate,ameliorate, any symptom caused by or resulting from an infection by aforeign agent. For the purposes of this disclosure a foreign agentincludes, but is not limited to, bacteria, parasites, viruses, andfungi.

Infections from organisms such as Group A Streptococcus (GAS,Streptococcus pyogenes) or Staphylococcus aureus range from superficialto invasive, and collectively represent a severe societal burden, onlyescalating with the increase of resistance to pharmaceutically derivedantibiotics (Jones, 2003; Carapetis et al., 2005; McCaig et al., 2006).Increasing the understanding of innate host-derived antimicrobialpeptides (AMPs) offers an alternative to the development of treatment ofsuch infections, as AMPs have retained the capacity to provideprotection against infections by GAS, S. aureus, and other microbes(Dorschner et al., 2001; Nizet et al., 2001; Di Nardo et al., 2008), andhave not lost their antimicrobial relevance as in the case of manypharmaceutical antibiotics.

A surprising recent revelation is that the AMPs that occupy the surfaceof the skin are made not only by the host cell, but also in prokaryoticorganisms that inhabit the host's epidermis. A large number of Grampositive bacteria such as Lactococcus, Streptococcus and Streptomycesspecies have been known to produce factors to inhibit other bacteria(Bastos et al., 2009). Proteinaceous factors produced by bacteria withbactericidal activity against the growth of similar or closely relatedbacterial strains are called bacteriocins. S. epidermidis, the dominantcommensal bacterium found in the skin microflora, produces various typesof bacteriocins. Most of these peptides are encoded in plasmids.Epidermin, Pep5 and epilancin K7 are the most characterized bacteriocinsisolated from S. epidermidis (Bastos et al., 2009). Because of theirpotential to kill pathogens in vitro, these bacteriocins may possess thecapacity to provide antimicrobial protection against pathogens on theskin surface. Research has shown unique peptides phenol-soluble modulin(PSM)γ and PSMδ produced by S. epidermidis could be beneficial to thehost and thus serve as additional AMPs on normal skin surface (Cogen etal., 2010). These peptides selectively exhibited bactericidal activityagainst skin pathogens, such as Staphylococcus aureus (S. aureus), GroupA Streptococcus (GAS) and Escherichia coli, whereas they are not activeagainst S. epidermidis. Moreover, inoculating PSMs on the mouse skinsurface reduced GAS but not the survival of S. epidermidis. Thisselective activity is likely to be an important part of a normalmicrobial defense strategy against colonization.

Staphylococcus epidermis (S. epidermidis) is a major constituent ofmicroflora on healthy human skin. Recent studies indicate that S.epidermidis protect human skin by preventing pathogenic infections byproducing phenol-soluble modulins (PSMs), which function asantimicrobial peptides. In addition, lipoteichoic acid produced by S.epidermidis benefits human skin by suppressing skin inflammation duringwound repair.

The disclosure provides a novel antimicrobial molecule from culturesupernatant of a clinically isolated strain of S. epidermidis (MO34).The predicted molecular formula is C₅H₅N₅O. The compound is referred toherein as “firmocidin.” Firmocidin exerts antimicrobial activitiesagainst group A streptococcus (GAS) and group B streptococcus (GBS) andStaphylococcus aureus (S. aureus), which are most common pathogens inhuman skin. Thus, firmocidin can be used as an antimicrobial treatmentto control skin infections.

NMR spectra data of firmocidin did not match any previouslycharacterized antimicrobial agents, suggesting firmocidin is a novelantimicrobial component (see, e.g., FIGS. 3, 9 and 10). In vitrobactericidal assays revealed that firmocidin showed antimicrobialactivity against GAS, GBS and Staphylococcus aureus, which are mostcommon pathogens in the human skin (see, e.g., FIG. 12). Most notably,firmocidin suppresses growth of methicillin-resistant S. aureus (MRSA),a strain that is highly resistant to some antibiotics (see, e.g., FIG.12).

Existing antibiotic therapies are non-specific bactericidals that maydisrupt the homeostasis of skin-resident microflora. However, firmocidindoes not affect the growth of S. epidermidis, which contributes normaldefense at the skin epithelium. Firmocidin also did not affect viabilityof human keratinocytes and sebocytes (see, e.g., FIG. 14). In addition,firmocidin is isolated from a microorganism residing in the normal skinmicroflora, suggesting low toxicity to the host. Thus, firmocidin can besafely used as a pathogen-specific antimicrobial therapy for skininfections.

The disclosure also provides a procedure for purification of firmocidinfrom culture supernatant of S. epidermidis (see e.g., FIG. 1). Thesample was purified by HPLC using a Taskgel NH₂-100 column (Tosoh) witha linear gradient of 5-20% water in acetonitrile (see e.g., FIG. 2). Apeak at the fraction #28 showed antimicrobial activity (see, e.g., FIG.2). High-resolution ES-MS analysis gave the molecular formula C₅H₅N₅O(calculated exact mass for [M+H]⁺=152.0567, observed mass=152.0567)(see, e.g., FIG. 3). Antimicrobial activity of the authentic compoundspossessing the molecular formula of C₅H₅N₅O were tested, but none ofthem showed antimicrobial activity against GAS (see e.g., FIG. 11).

According to the preliminary data from NMR structural determination, theNMR spectra for firmocidin did not match to any of authenticantimicrobial agents. This suggests that firmocidin is a novelantimicrobial compound that suppresses the growth of GAS, GBS,methicillin-sensitive S. aureus and MRSA, but not S. epidermidis (see,e.g., FIG. 12) and Gram negative bacteria. Its antimicrobial activitywas stronger than benzoyl peroxide (BPO), which is a frequently usedtopical medication. Furthermore, kinetic study of antimicrobial activityshowed that firmocidin exhibits bactericidal activity against GAS andbacteriostatic activity against GBS and S. aureus (see, e.g., FIG. 13),but not against S. epidermidis. However, firmocidin did not affect theviabilities of human keratinocytes and sebocytes (see, e.g., FIG. 14),suggesting low toxicity to the host. Thus, firmocidin ispathogen-specific and safe antimicrobial therapy for infections arisingfrom GAS, GBS and S. aureus. Firmocidin, however, is likely to exertsimilar antimicrobial activity against other pathogens.

Methods and compositions useful for treatment of microbial infectionsare provided. In one embodiment the disclosure provides compositions andmethods useful for treating a microbial infection wherein the methodsand compositions comprise firmocidin, a derivative or salt thereof. Forexample, methods and compositions useful for the treatment of S. aureusinfections, including those produced by methicillin- andvancomycin-resistant strains, are provided. The methods and compositionsof the disclosure can be used alone or in combination with traditionalantimicrobials and antibiotics to treat such infections. In addition,the methods and compositions disclosed herein can be used in settingssuch as foreign-body, catheter or endovascular infections, chronicosteomyelitis, hospital acquired or post-operative infections, recurrentskin infections, or for S. aureus infections in the immunocompromisedhost.

In a certain embodiment, the disclosure provides for a compound that iscomprised of one or more ring structures that is comprised of at least 5carbon atoms, at least 5 hydrogen atoms, at least 5 nitrogen atoms, andat least 1 oxygen atom, wherein the ring structures are optionallysubstituted and selected from the group comprising cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heterocycle, and mixed ring system. Inanother embodiment, the disclosure provides for a compound that iscomprised of one or more ring structures that is comprised of 5 carbonatoms, 5 hydrogen atoms, 5 nitrogen atoms, and 1 oxygen atom, whereinthe ring structures are optionally substituted and selected from thegroup comprising cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heterocycle, and mixed ring system. It should be understood for aheterocycle-, and a mixed ring system-based compound that while it maybe preferable that one or more hetero-ring atoms is an N and/or O, thedisclosure also provides for additional heteroatoms, including, but notlimited to, N, O, S, Si, Al, B, and P. Likewise, it should also beunderstood for a compound comprised of a cycloalkyl, a cycloalkenyl, acycloalkynyl, or an aryl, that while it may be preferable that theserings be substituted with either one or more oxygen containingfunctional groups, nitrogen containing functional groups, or acombination thereof, the disclosure also provides for a compoundcomprised of a cycloalkyl, a cycloalkenyl, a cycloalkynyl, or an aryl,being substituted with functional groups which do not contain oxygenand/or nitrogen atoms.

In a particular embodiment, the disclosure provides for a compoundhaving structural Formula I

wherein,

X¹-X¹⁰ are each independently either a C, N or O;

R¹-R¹⁸ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence.

In an additional embodiment, the disclosure provides for a compoundhaving structural Formula I:

wherein,

X¹-X¹⁰ are each independently either a C, N or O;

R¹-R¹⁸ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In a further embodiment, the disclosure provides for a compound havingstructural Formula I(a)

wherein,

X¹-X¹⁰ are each independently either a C, N or O;

R¹, R³, R⁷, R⁹, R¹¹, R¹³, R¹⁵, and R¹⁷ are each independently selectedfrom the group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In a further embodiment, the disclosure provides for a compound havingstructural Formula I(a):

wherein,

X¹-X¹⁰ are each independently either a C, N or O;

R¹, R³, R⁷, R⁹, R¹¹, R¹³, R¹⁵, and R¹⁷ are each independently selectedfrom the group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has 5 carbon atoms, 5 hydrogen atoms, 5 nitrogenatoms, and 1 oxygen atom.

In yet a further embodiment, the disclosure provides for a compoundselected from the group comprising

In a particular embodiment, the disclosure provides a compound havingstructural Formula II

wherein,

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹-R³⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence.

In another embodiment, the disclosure provides a compound havingstructural Formula II

wherein,

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹-R³⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In yet another embodiment, the disclosure provides for a compound havingstructural Formula II(a):

wherein,

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹, R²³, R²⁵, R²⁷, R²⁹, R³¹, and R³³ are each independently selectedfrom the group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In a further embodiment, the disclosure provides for a compound havingstructural Formula II(a):

wherein,

X¹¹-X¹⁹ are each independently either a C, N or O;

R¹⁹, R²³, R²⁵, R²⁷, R²⁹, R³¹, and R³³ are each independently selectedfrom the group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has 5 carbon atoms, 5 hydrogen atoms, 5 nitrogenatoms, and 1 oxygen atom.

In a certain embodiment, the disclosure provides for a compound ofselected from the group comprising

In a particular embodiment, the disclosure provides for a compoundhaving structural Formula III:

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵-R⁵⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence.

In a particular embodiment, the disclosure provides for a compoundhaving structural Formula III:

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵-R⁵⁴ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In a further embodiment, the disclosure provides for a compound havingstructural Formula III(a)

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵, R³⁷, R⁴¹, R⁴³, R⁴⁵, R⁴⁷, R⁴⁹, R⁵¹, and R⁵³, are each independentlyselected from the group comprising H, D, optionally substituted(C₁-C₂)alkyl, optionally substituted (C₁-C₂)alkenyl, optionallysubstituted (C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In yet a further embodiment, the disclosure provides for a compoundhaving structural Formula III(a):

wherein,

X²⁰-X³⁰ are each independently either a C, N or O;

R³⁵, R³⁷, R⁴¹, R⁴³, R⁴⁵, R⁴⁷, R⁴⁹, R⁵¹, and R⁵³, are each selected fromthe group comprising H, D, optionally substituted (C₁-C₂)alkyl,optionally substituted (C₁-C₂)alkenyl, optionally substituted(C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has 5 carbon atoms, 5 hydrogen atoms, 5 nitrogenatoms, and 1 oxygen atom.

In yet a further embodiment, the disclosure provides for a compoundhaving the structure of

In a particular embodiment, the disclosure provides for a compoundhaving structural Formula IV:

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵-R⁶⁹ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₆)alkyl, optionally substituted(C₁-C₆)alkenyl, optionally substituted (C₁-C₆)alkynyl, optionallysubstituted hetero-(C₁-C₆)alkyl, hetero-(C₁-C₆)alkenyl, optionallysubstituted hetero-(C₁-C₆)alkynyl, halogen, hydroxyl, ketone, aldehyde,acyl halide, carbonate, carboxylic acid, ester, hydroperoxide, peroxide,ether, hemiacetal, hemiketal, acetal, orthoester, orthocarbonate ester,amide, amine, imine, imide, azide, diimide, cyanate, nitrate, nitrile,nitro, nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinicacid, sulfonic acid, thicyanate, thione, thial, phosphine, phosphonicacid, phosphate, phosphodiester, boronic acid, boronic ester, and noatom if bound to X that has reached its maximum valence.

In another embodiment, the disclosure provides a compound havingstructural Formula IV:

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵-R⁶⁹ are each independently selected from the group comprising H, D,optionally substituted (C₁-C₂)alkyl, optionally substituted(C₁-C₂)alkenyl, optionally substituted (C₁-C₂)alkynyl, optionallysubstituted hetero-(C₁-C₂)alkyl, hetero-(C₁-C₂)alkenyl, optionallysubstituted hetero-(C₁-C₂)alkynyl, hydroxyl, ketone, aldehyde, ester,ether, amide, amine, imine, imide, nitrate, nitrile, nitro, nitroso, andno atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In yet another embodiment, the disclosure provides for a compound havingstructural Formula IV(a):

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵, R⁵⁷, R⁵⁹, R⁶¹, R⁶³, R⁶⁵, R⁶⁷, and R⁶⁹ are each independentlyselected from the group comprising H, D, optionally substituted(C₁-C₂)alkyl, optionally substituted (C₁-C₂)alkenyl, optionallysubstituted (C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has at least 5 carbon atoms, at least 5 hydrogenatoms, at least 5 nitrogen atoms, and at least 1 oxygen atom.

In a further embodiment, the disclosure provides for a compound havingstructural Formula IV(a):

wherein,

X³¹-X³⁸ are each independently either a C, N or O;

R⁵⁵, R⁵⁷, R⁵⁹, R⁶¹, R⁶³, R⁶⁵, R⁶⁷, and R⁶⁹ are each independentlyselected from the group comprising H, D, optionally substituted(C₁-C₂)alkyl, optionally substituted (C₁-C₂)alkenyl, optionallysubstituted (C₁-C₂)alkynyl, optionally substituted hetero-(C₁-C₂)alkyl,hetero-(C₁-C₂)alkenyl, optionally substituted hetero-(C₁-C₂)alkynyl,hydroxyl, ketone, aldehyde, carbonate, amine, imine, nitrile, nitroso,and no atom if bound to X that has reached its maximum valence; and

wherein the compound has 5 carbon atoms, 5 hydrogen atoms, 5 nitrogenatoms, and 1 oxygen atom.

In yet a further embodiment, the disclosure provides for a compoundhaving the structure of

In yet a further embodiment, a compound disclosed herein that containsan acidic or basic moiety may also be disclosed as a pharmaceuticallyacceptable salt (See, Berge et al., J. Pharma. Sci. 1977, 66, 1-19; and“Handbook of Pharmaceutical Salts, Properties, and Use,” Stah andWermuth, Ed.; Wiley-VCH and VHCA, Zurich, 2002).

Suitable acids for use in the preparation of pharmaceutically acceptablesalts, include, but are not limited to, aceptic acid, 2,2-dichloroaceticacid, acylated amino acids, adipic acid, alginic acid, ascorbic acid,L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoicacid, boric acid, (+)-camphoric acid, camphorsulfonic acid,(+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylicacid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamicacid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonicacid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid,galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid,D-glucuronic acid, L-glutamic acid, α-oxo-glutaric acid, glycolic acid,hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid,(+)-L-lactic acid, (+/−)-DL-lactic acid, lactobionic acid, lauric acid,maleic acid, (−)-L-malic acid, malonic acid, (+/−)-DL-mandelic acid,methanesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 1-hydroxy-2-napthoic acid, nicotinicacid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid,pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid,saccharic acid, salicyclic acid, 4-amino-salicylic acid, sebacic acid,stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaricacid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, andvaleric acid.

Suitable acids for use in the preparation of pharmaceutically acceptablesalts, include, but are not limited to, inorganic bases, such asmagnesium hydroxide, calcium hydroxide, potassium hydroxide, zinchydroxide, or sodium hydroxide; and organic bases, such as primary,secondary, tertiary, and quaternary, aliphatic and aromatic amines,including L-arginine, benethamine, benzathine, choline, deanol,diethanolamine, diethylamine, dimethylamine, dipropylamine,diisopropylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylamine,ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine,1H-imidazole, L-Lysine, morpholine, 4-(2-hydroxyethyl)-morpholine,methylamine, piperidine, piperazine, propylamine, pyrrolidine,1-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline,isoquinoline, secondary amines, triethanolamine, trimethylamine,triethylamine, N-methyl-D-glucamine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and tromethamine.

Pharmaceutically acceptable salts comprise pharmaceutically-acceptableanions and/or cations. Pharmaceutically-acceptable cations include amongothers, alkali metal cations (e.g., Li+, Na+, K+), alkaline earth metalcations (e.g., Ca²⁺, Mg²⁺), non-toxic heavy metal cations and ammonium(NH₄ ⁺) and substituted ammonium (N(R′)₄ ⁺, where R′ is hydrogen, alkyl,or substituted alkyl, i.e., including, methyl, ethyl, or hydroxyethyl,specifically, trimethyl ammonium, triethyl ammonium, and triethanolammonium cations). Pharmaceutically-acceptable anions include amongother halides (e.g., Cl—, Br—), sulfate, acetates (e.g., acetate,trifluoroacetate), ascorbates, aspartates, benzoates, citrates, andlactate.

A compound disclosed herein may also have a prodrug form. A prodrug is afunctional derivative of the compound disclosed herein and is readilyconvertible into the parent compound in vivo. Prodrugs are often usefulbecause, in some situations, they may be easier to administer than theparent compound. They may, for instance, be bio-available by oraladministration whereas the parent compound is not. The prodrug may alsohave enhanced solubility in pharmaceutical compositions over the parentcompound. A prodrug may be converted into the parent drug by variousmechanisms, including enzymatic processes and metabolic hydrolysis.Various examples and forms of prodrugs are well known in the art.Examples of prodrugs are found, inter alia, in Design of Prodrugs,edited by H. Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol.42, at pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985);A Textbook of Drug Design and Development, edited by Krosgaard-Larsenand H. Bundgaard, Chapter 5, “Design and Application of Prodrugs,” by H.Bundgaard, at pp. 113-191, 1991); H. Bundgaard, Advanced Drug DeliveryReviews, Vol. 8, p. 1-38 (1992); H. Bundgaard, et al., Journal ofPharmaceutical Sciences, Vol. 77, p. 285 (1988); and Nogrady (1985)Medicinal Chemistry A Biochemical Approach, Oxford University Press, NewYork, pages 388-392). In a specific example, if a parent compounddisclosed herein has a hydroxyl group, this hydroxyl group may beconverted to an ester in attempts to increase bioavailability,solubility, injection site pain relief, elimination of an unpleasanttaste, decreased toxicity, decreased metabolic inactivation, increasedchemical stability, and/or prolonged or shortened action of the hydroxylcontaining parent compound. In another specific example, if a parentcompound disclosed herein has an amine group, this amine group may beconverted to a Schiff base in attempts to increase bioavailability,solubility, injection site pain relief, elimination of an unpleasanttaste, decreased toxicity, decreased metabolic inactivation, increasedchemical stability, and/or prolonged or shortened action of the hydroxylcontaining parent compound.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, can be in a form suitable for administration to a subjectusing carriers, excipients, and additives or auxiliaries. Frequentlyused carriers or auxiliaries include magnesium carbonate, titaniumdioxide, lactose, mannitol and other sugars, talc, milk protein,gelatin, starch, vitamins, cellulose and its derivatives, animal andvegetable oils, polyethylene glycols and solvents, such as sterilewater, alcohols, glycerol, and polyhydric alcohols. Intravenous vehiclesinclude fluid and nutrient replenishers. Preservatives includeantimicrobial, chelating agents, and inert gases. Other pharmaceuticallyacceptable carriers include aqueous solutions, non-toxic excipients,including salts, preservatives, buffers and the like, as described, forinstance, in Remington's Pharmaceutical Sciences, 15th ed., Easton: MackPublishing Co., 1405-1412, 1461-1487 (1975), and The National FormularyXIV, 14th ed., Washington: American Pharmaceutical Association (1975),the contents of which are hereby incorporated by reference. The pH andexact concentration of the various components of the pharmaceuticalcomposition are adjusted according to routine skills in the art. SeeGoodman and Gilman's, The Pharmacological Basis for Therapeutics (7thed.).

The pharmaceutical compositions according to the disclosure may beadministered locally or systemically. A “therapeutically effective dose”is the quantity of an agent according to the disclosure necessary toprevent, to cure, or at least partially arrest the symptoms of aninfection by a foreign agent. Amounts effective for this use will, ofcourse, depend on the severity of the disease and the weight and generalstate of the subject. Typically, dosages used in vitro may provideuseful guidance in the amounts useful for in situ administration of thepharmaceutical composition, and animal models may be used to determineeffective dosages for treatment of infections. Various considerationsare described, e.g., in Langer, Science, 249: 1527, (1990); Gilman etal. (eds.) (1990), each of which is herein incorporated by reference.

As used herein, “administering a therapeutically effective amount” isintended to include methods of giving or applying a pharmaceuticalcomposition of the disclosure to a subject that allow the composition toperform its intended therapeutic function. The therapeutically effectiveamounts will vary according to factors, such as the degree of infectionin a subject, the age, sex, and weight of the individual. Dosage regimacan be adjusted to provide the optimum therapeutic response. Forexample, several divided doses can be administered daily or the dose canbe proportionally reduced as indicated by the exigencies of thetherapeutic situation.

Thus, a “pharmaceutically acceptable carrier” is intended to includesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the pharmaceutical composition, use thereof in thetherapeutic compositions and methods of treatment is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The principal pharmaceutical composition is compounded for convenientand effective administration in effective amounts with a suitablepharmaceutically acceptable carrier in an acceptable dosage unit. In thecase of compositions containing supplementary active ingredients, thedosages are determined by reference to the usual dose and manner ofadministration of the said ingredients.

The disclosure provides for a compound disclosed herein, derivative oranalog thereof, including pharmaceutical salt forms and prodrug forms,can be administered to any host, including a human or non-human animal,in an amount effective to inhibit the growth, spread or infection by aforeign agent. In one embodiment, the administration results in theinhibition of growth of a bacterium, virus, parasite and/or fungus.Thus, the methods and compositions are useful as antimicrobial agents.

Any of a variety of art-known methods can be used to administer acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, either alone or used incombination with one or more other therapeutic agents. For example,administration can be parenterally by injection or by gradual infusionover time. The agent(s) can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity, byinhalation, or transdermally.

The pharmaceutical composition can be administered in a convenientmanner, such as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, thepharmaceutical composition can be coated with a material to protect thepharmaceutical composition from the action of enzymes, acids, and othernatural conditions that may inactivate the pharmaceutical composition.The pharmaceutical composition can also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof, and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

In another embodiment, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, can be formulated eitheralone or in combination with one or more additional therapeutic agents,including, but not limited to, antibiotics, antifungal-agents,anti-pruritics, analgesics, and/or antiviral agents, for topicaladministration. The topical administration, as used herein, include(intra)dermal, conjunctival, intracorneal, intraocular, ophthalmic,auricular, transdermal, nasal, vaginal, uretheral, respiratory, andrectal administration. Such topical formulations are useful in treatingor inhibiting infections of the eye, skin, and mucous membranes (e.g.,mouth, vagina). Examples of formulations in the market place includetopical lotions, creams, soaps, wipes, and the like.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated in any dosage form that is suitable fortopical administration for local or systemic effect, includingemulsions, solutions, suspensions, creams, gels, hydrogels, ointments,dusting powders, dressings, elixirs, lotions, suspensions, tinctures,pastes, foams, films, aerosols, irrigations, sprays, suppositories,bandages, dermal patches. The topical formulation comprising a compounddisclosed herein may also comprise liposomes, micelles, microspheres,nanosystems, and mixtures thereof.

Pharmaceutically acceptable carriers and excipients suitable for use inthe topical formulations disclosed herein include, but are not limitedto, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles,antimicrobial agents or preservatives against the growth ofmicroorganisms, stabilizers, solubility enhancers, isotonic agents,buffering agents, antioxidants, local anesthetics, suspending anddispersing agents, wetting or emulsifying agents, complexing agents,sequestering or chelating agents, penetration enhancers,cryoprotectants, lyoprotectants, thickening agents, and inert gases.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may also be administered topically by electroporation,iontophoresis, phonophoresis, sonophoresis and microneedle orneedle-free injection, such as POWDERJECT™ (Chiron Corp., Emeryville,Calif.), and BIOJECT™ (Bioject Medical Technologies Inc., Tualatin,Oreg.).

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated in the forms of ointments, creams,sprays and gels. Suitable ointment vehicles include oleaginous orhydrocarbon vehicles, including such as lard, benzoinated lard, oliveoil, cottonseed oil, and other oils, white petrolatum; emulsifiable orabsorption vehicles, such as hydrophilic petrolatum, hydroxystearinsulfate, and anhydrous lanolin; water-removable vehicles, such ashydrophilic ointment; water-soluble ointment vehicles, includingpolyethylene glycols of varying molecular weight; emulsion vehicles,either water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions,including cetyl alcohol, glyceryl monostearate, lanolin, and stearicacid (see, Remington: The Science and Practice of Pharmacy, supra).These vehicles are emollient but generally require addition ofantioxidants and preservatives.

Suitable cream base can be oil-in-water or water-in-oil. Cream vehiclesmay be water-washable, and contain an oil phase, an emulsifier, and anaqueous phase. The oil phase is also called the “internal” phase, whichis generally comprised of petrolatum and a fatty alcohol such as cetylor stearyl alcohol. The aqueous phase usually, although not necessarily,exceeds the oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation may be a nonionic, anionic, cationic,or amphoteric surfactant.

Gels are semisolid, suspension-type systems. Single-phase gels containorganic macromolecules distributed substantially uniformly throughoutthe liquid carrier. Suitable gelling agents include crosslinked acrylicacid polymers, such as carbomers, carboxypolyalkylenes, Carbopol®;hydrophilic polymers, such as polyethylene oxides,polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, and methylcellulose; gums, such as tragacanth and xanthangum; sodium alginate; and gelatin. In order to prepare a uniform gel,dispersing agents such as alcohol or glycerin can be added, or thegelling agent can be dispersed by trituration, mechanical mixing, and/orstirring.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be administered rectally, urethrally, vaginally, orperivaginally in the forms of suppositories, pessaries, bougies,poultices or cataplasm, pastes, powders, dressings, creams, plasters,contraceptives, ointments, solutions, emulsions, suspensions, tampons,gels, foams, sprays, or enemas. These dosage forms can be manufacturedusing conventional processes as described in Remington: The Science andPractice of Pharmacy, supra.

Rectal, urethral, and vaginal suppositories are solid bodies forinsertion into body orifices, which are solid at ordinary temperaturesbut melt or soften at body temperature to release the activeingredient(s) inside the orifices. Pharmaceutically acceptable carriersutilized in rectal and vaginal suppositories include bases or vehicles,such as stiffening agents, which produce a melting point in theproximity of body temperature, when formulated with the pharmaceuticalcompositions disclosed herein; and antioxidants as described herein,including bisulfite and sodium metabisulfite. Suitable vehicles include,but are not limited to, cocoa butter (theobroma oil), glycerin-gelatin,carbowax (polyoxyethylene glycol), spermaceti, paraffin, white andyellow wax, and appropriate mixtures of mono-, di- and triglycerides offatty acids, hydrogels, such as polyvinyl alcohol, hydroxyethylmethacrylate, polyacrylic acid; glycerinated gelatin. Combinations ofthe various vehicles may be used. Rectal and vaginal suppositories maybe prepared by the compressed method or molding. The typical weight of arectal and vaginal suppository is about 2 to about 3 g.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be administered ophthalmically in the forms ofsolutions, suspensions, ointments, emulsions, gel-forming solutions,powders for solutions, gels, ocular inserts, and implants. Apharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be administered intranasally or by inhalation to therespiratory tract. A pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, may be formulated in theform of an aerosol or solution for delivery using a pressurizedcontainer, pump, spray, atomizer, such as an atomizer usingelectrohydrodynamics to produce a fine mist, or nebulizer, alone or incombination with a suitable propellant, such as1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. Apharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may also be formulated as a dry powder for insufflation,alone or in combination with an inert carrier such as lactose orphospholipids; and nasal drops. For intranasal use, the powder maycomprise a bioadhesive agent, including chitosan or cyclodextrin.

Solutions or suspensions for use in a pressurized container, pump,spray, atomizer, or nebulizer may be formulated to contain ethanol,aqueous ethanol, or a suitable alternative agent for dispersing,solubilizing, or extending release of the active ingredient disclosedherein, a propellant as solvent; and/or a surfactant, such as sorbitantrioleate, oleic acid, or an oligolactic acid.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be micronized to a size suitable for delivery byinhalation, such as about 50 micrometers or less, or about 10micrometers or less. Particles of such sizes may be prepared using acomminuting method known to those skilled in the art, such as spiral jetmilling, fluid bed jet milling, supercritical fluid processing to formnanoparticles, high pressure homogenization, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the pharmaceuticalcompositions disclosed herein; a suitable powder base, such as lactoseor starch; and a performance modifier, such as 1-leucine, mannitol, ormagnesium stearate. The lactose may be anhydrous or in the form of themonohydrate. Other suitable excipients or carriers include dextran,glucose, maltose, sorbitol, xylitol, fructose, sucrose, and trehalose. Apharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, for inhaled/intranasal administration may furthercomprise a suitable flavor, such as menthol and levomenthol, orsweeteners, such as saccharin or saccharin sodium.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, for topical administration may be formulated to beimmediate release or modified release, including delayed-, sustained-,pulsed-, controlled-, targeted, and programmed release.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated into liposomes to reduce toxicity orincrease bioavailability. Other methods for delivery include oralmethods that entail encapsulation of the in microspheres or proteinoids,aerosol delivery (e.g., to the lungs), or transdermal delivery (e.g., byiontophoresis or transdermal electroporation). Other methods ofadministration will be known to those skilled in the art.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated as a modified release dosage form. Asused herein, the term “modified release” refers to a dosage form inwhich the rate or place of release of the active ingredient(s) isdifferent from that of an immediate dosage form when administered by thesame route. Modified release dosage forms include delayed-, extended-,prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-,targeted-, programmed-release, and gastric retention dosage forms. Thepharmaceutical compositions in modified release dosage forms can beprepared using a variety of modified release devices and methods knownto those skilled in the art, including, but not limited to, matrixcontrolled release devices, osmotic controlled release devices,multiparticulate controlled release devices, ion-exchange resins,enteric coatings, multilayered coatings, microspheres, liposomes, andcombinations thereof. The release rate of the active ingredient(s) canalso be modified by varying the particle sizes and polymorphorism of theactive ingredient(s). Examples of modified release include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566;5,739,108; 5,891,474; 5,922,356; 5,972,891; 5,980,945; 5,993,855;6,045,830; 6,087,324; 6,113,943; 6,197,350; 6,248,363; 6,264,970;6,267,981; 6,376,461; 6,419,961; 6,589,548; 6,613,358; and 6,699,500.

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, that is formulated in a modified release dosage form maybe fabricated using a matrix controlled release device (see, Takada etal in “Encyclopedia of Controlled Drug Delivery,” Vol. 2, Mathiowitzed., Wiley, 1999).

In one embodiment, the pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, in a modified releasedosage form is formulated using an erodible matrix device, which iswater-swellable, erodible, or soluble polymers, including syntheticpolymers, and naturally occurring polymers and derivatives, such aspolysaccharides and proteins.

Materials useful in forming an erodible matrix include, but are notlimited to, chitin, chitosan, dextran, and pullulan; gum agar, gumarabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gumghatti, guar gum, xanthan gum, and scleroglucan; starches, such asdextrin and maltodextrin; hydrophilic colloids, such as pectin;phosphatides, such as lecithin; alginates; propylene glycol alginate;gelatin; collagen; and cellulosics, such as ethyl cellulose (EC),methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC,hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), celluloseacetate (CA), cellulose propionate (CP), cellulose butyrate (CB),cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methylcellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetatetrimellitate (HPMCAT), and ethylhydroxy ethylcellulose (EHEC); polyvinylpyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acidesters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acidor methacrylic acid (EUDRAGIT, Rohm America, Inc., Piscataway, N.J.);poly(2-hydroxyethyl-methacrylate); polylactides; copolymers ofL-glutamic acid and ethyl-L-glutamate; degradable lactic acid-glycolicacid copolymers; poly-D-(−)-3-hydroxybutyric acid; and other acrylicacid derivatives, such as homopolymers and copolymers ofbutylmethacrylate, methylmethacrylate, ethylmethacrylate, ethylacrylate,(2-dimethylaminoethyl)methacrylate, and(trimethylaminoethyl)methacrylate chloride.

In further embodiments, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated with anon-erodible matrix device. The active ingredient(s) is dissolved ordispersed in an inert matrix and is released primarily by diffusionthrough the inert matrix once administered. Materials suitable for useas a non-erodible matrix device included, but are not limited to,insoluble plastics, such as polyethylene, polypropylene, polyisoprene,polyisobutylene, polybutadiene, polymethylmethacrylate,polybutylmethacrylate, chlorinated polyethylene, polyvinylchloride,methyl acrylate-methyl methacrylate copolymers, ethylene-vinylacetatecopolymers, ethylene/propylene copolymers, ethylene/ethyl acrylatecopolymers, vinylchloride copolymers with vinyl acetate, vinylidenechloride, ethylene and propylene, ionomer polyethylene terephthalate,butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, polyvinyl chloride, plasticizednylon, plasticized polyethyleneterephthalate, natural rubber, siliconerubbers, polydimethylsiloxanes, silicone carbonate copolymers;hydrophilic polymers, such as ethyl cellulose, cellulose acetate,crospovidone, and cross-linked partially hydrolyzed polyvinyl acetate;and fatty compounds, such as carnauba wax, microcrystalline wax, andtriglycerides.

In a matrix controlled release system, the desired release kinetics canbe controlled, for example, via the polymer type employed, the polymerviscosity, the particle sizes of the polymer and/or the activeingredient(s), the ratio of the active ingredient(s) versus the polymer,and other excipients or carriers in the compositions.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, in a modified release dosage form may be prepared bymethods known to those skilled in the art, including direct compression,dry or wet granulation followed by compression, melt-granulationfollowed by compression.

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, in a modified release dosage form may be fabricated usingan osmotic controlled release device, including one-chamber system,two-chamber system, asymmetric membrane technology (AMT), and extrudingcore system (ECS). In general, such devices have at least twocomponents: (a) the core which contains the active ingredient(s) and (b)a semipermeable membrane with at least one delivery port, whichencapsulates the core. The semipermeable membrane controls the influx ofwater to the core from an aqueous environment of use so as to cause drugrelease by extrusion through the delivery port(s).

In addition to the active ingredient(s), the core of the osmotic deviceoptionally includes an osmotic agent, which creates a driving force fortransport of water from the environment of use into the core of thedevice. One class of osmotic agents water-swellable hydrophilicpolymers, which are also referred to as “osmopolymers” and “hydrogels,”including, but not limited to, hydrophilic vinyl and acrylic polymers,polysaccharides such as calcium alginate, polyethylene oxide (PEO),polyethylene glycol (PEG), polypropylene glycol (PPG),poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic)acid, polyvinylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol(PVA), PVA/PVP copolymers, PVA/PVP copolymers with hydrophobic monomerssuch as methyl methacrylate and vinyl acetate, hydrophilic polyurethanescontaining large PEO blocks, sodium croscarmellose, carrageenan,hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC),hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) andcarboxyethyl, cellulose (CEC), sodium alginate, polycarbophil, gelatin,xanthan gum, and sodium starch glycolate.

The other class of osmotic agents are osmogens, which are capable ofimbibing water to affect an osmotic pressure gradient across the barrierof the surrounding coating. Suitable osmogens include, but are notlimited to, inorganic salts, such as magnesium sulfate, magnesiumchloride, calcium chloride, sodium chloride, lithium chloride, potassiumsulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithiumsulfate, potassium chloride, and sodium sulfate; sugars, such asdextrose, fructose, glucose, inositol, lactose, maltose, mannitol,raffinose, sorbitol, sucrose, trehalose, and xylitol; organic acids,such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleicacid, sebacic acid, sorbic acid, adipic acid, edetic acid, glutamicacid, p-tolunesulfonic acid, succinic acid, and tartaric acid; urea; andmixtures thereof.

Osmotic agents of different dissolution rates may be employed toinfluence how rapidly the active ingredient(s) is initially deliveredfrom the dosage form. For example, amorphous sugars, such as MannogemeEZ (SPI Pharma, Lewes, Del.) can be used to provide faster deliveryduring the first couple of hours to promptly produce the desiredtherapeutic effect, and gradually and continually release of theremaining amount to maintain the desired level of therapeutic orprophylactic effect over an extended period of time. In this case, theactive ingredient(s) is released at such a rate to replace the amount ofthe active ingredient metabolized and excreted.

The core may also include a wide variety of other excipients andcarriers as described herein to enhance the performance of the dosageform or to promote stability or processing.

Materials useful in forming the semipermeable membrane include variousgrades of acrylics, vinyls, ethers, polyamides, polyesters, andcellulosic derivatives that are water-permeable and water-insoluble atphysiologically relevant pHs, or are susceptible to being renderedwater-insoluble by chemical alteration, such as crosslinking. Examplesof suitable polymers useful in forming the coating, include plasticized,unplasticized, and reinforced cellulose acetate (CA), cellulosediacetate, cellulose triacetate, CA propionate, cellulose nitrate,cellulose acetate butyrate (CAB), CA ethyl carbamate, CAP, CA methylcarbamate, CA succinate, cellulose acetate trimellitate (CAT), CAdimethylaminoacetate, CA ethyl carbonate, CA chloroacetate, CA ethyloxalate, CA methyl sulfonate, CA butyl sulfonate, CA p-toluenesulfonate, agar acetate, amylose triacetate, beta glucan acetate, betaglucan triacetate, acetaldehyde dimethyl acetate, triacetate of locustbean gum, hydroxlated ethylene-vinylacetate, EC, PEG, PPG, PEG/PPGcopolymers, PVP, HEC, HPC, CMC, CMEC, HPMC, HPMCP, HPMCAS, HPMCAT,poly(acrylic) acids and esters and poly-(methacrylic) acids and estersand copolymers thereof, starch, dextran, dextrin, chitosan, collagen,gelatin, polyalkenes, polyethers, polysulfones, polyethersulfones,polystyrenes, polyvinyl halides, polyvinyl esters and ethers, naturalwaxes, and synthetic waxes.

Semipermeable membrane may also be a hydrophobic microporous membrane,wherein the pores are substantially filled with a gas and are not wettedby the aqueous medium but are permeable to water vapor, as disclosed inU.S. Pat. No. 5,798,119. Such hydrophobic but water-vapor permeablemembrane are typically composed of hydrophobic polymers such aspolyalkenes, polyethylene, polypropylene, polytetrafluoroethylene,polyacrylic acid derivatives, polyethers, polysulfones,polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidenefluoride, polyvinyl esters and ethers, natural waxes, and syntheticwaxes.

The delivery port(s) on the semipermeable membrane may be formedpost-coating by mechanical or laser drilling. Delivery port(s) may alsobe formed in situ by erosion of a plug of water-soluble material or byrupture of a thinner portion of the membrane over an indentation in thecore. In addition, delivery ports may be formed during coating process,as in the case of asymmetric membrane coatings of the type disclosed inU.S. Pat. Nos. 5,612,059 and 5,698,220.

The total amount of the active ingredient(s) released and the releaserate can substantially by modulated via the thickness and porosity ofthe semipermeable membrane, the composition of the core, and the number,size, and position of the delivery ports.

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, in an osmotic controlled-release dosage form may furthercomprise additional conventional excipients or carriers as describedherein to promote performance or processing of the formulation.

The osmotic controlled-release dosage forms can be prepared according toconventional methods and techniques known to those skilled in the art(see, Remington: The Science and Practice of Pharmacy, supra; Santus andBaker, J. Controlled Release 1995, 35, 1-21; Verma et al., DrugDevelopment and Industrial Pharmacy 2000, 26, 695-708; Verma et al., J.Controlled Release 2002, 79, 7-27).

In a certain embodiment, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated as AMTcontrolled-release dosage form, which comprises an asymmetric osmoticmembrane that coats a core comprising the active ingredient(s) and otherpharmaceutically acceptable excipients or carriers. See, U.S. Pat. No.5,612,059 and WO 2002/17918. The AMT controlled-release dosage forms canbe prepared according to conventional methods and techniques known tothose skilled in the art, including direct compression, dry granulation,wet granulation, and a dip-coating method.

In a particular embodiment, a pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated as ESCcontrolled-release dosage form, which comprises an osmotic membrane thatcoats a core comprising the active ingredient(s), a hydroxylethylcellulose, and other pharmaceutically acceptable excipients or carriers.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be administered parenterally by injection, infusion,or implantation, for local or systemic administration. Parenteraladministration, as used herein, include intravenous, intraarterial,intraperitoneal, intrathecal, intraventricular, intraurethral,intrasternal, intracranial, intramuscular, intrasynovial, andsubcutaneous administration.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may be formulated in any dosage forms that are suitablefor parenteral administration, including solutions, suspensions,emulsions, micelles, liposomes, microspheres, nanosystems, and solidforms suitable for solutions or suspensions in liquid prior toinjection. Such dosage forms can be prepared according to conventionalmethods known to those skilled in the art of pharmaceutical science(see, Remington: The Science and Practice of Pharmacy, supra).

The pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, may include one or more pharmaceutically acceptablecarriers and excipients, including, but not limited to, aqueousvehicles, water-miscible vehicles, non-aqueous vehicles, antibacterialagents or preservatives against the growth of microorganisms,stabilizers, solubility enhancers, isotonic agents, buffering agents,antioxidants, local anesthetics, suspending and dispersing agents,wetting or emulsifying agents, complexing agents, sequestering orchelating agents, cryoprotectants, lyoprotectants, thickening agents, pHadjusting agents, and inert gases.

Preparations for parenteral administration of a pharmaceuticalcomposition comprising a compound disclosed herein, a derivative oranalog thereof, including pharmaceutical salt forms and prodrug formsinclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Suitable aqueous vehicles include, but are not limited to,water, saline, physiological saline or phosphate buffered saline (PBS),sodium chloride injection, Ringers injection, isotonic dextroseinjection, sterile water injection, dextrose, lactated Ringersinjection, alcoholic/aqueous solutions, and emulsions or suspensions.Non-aqueous vehicles include, but are not limited to, injectable organicesters such as ethyl oleate, and fixed oils of vegetable origin, castoroil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils,hydrogenated soybean oil, and medium-chain triglycerides of coconut oil,palm seed oil. Water-miscible vehicles include, but are not limited to,ethanol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethyleneglycol 300 and polyethylene glycol 400), propylene glycol, glycerin,N-methyl-2-pyrrolidone, dimethylacetamide, and dimethylsulfoxide.Examples of parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, andfixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives such as,other antibacterials, anti-oxidants, cheating agents, inert gases andthe like also can be included.

Suitable antibacterial agents or preservatives include, but are notlimited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol,methyl and propyl p-hydroxybenzates, thimerosal, benzalkonium chloride,benzethonium chloride, methyl- and propyl-parabens, and sorbic acid.Suitable isotonic agents include, but are not limited to, sodiumchloride, glycerin, and dextrose. Suitable buffering agents include, butare not limited to, phosphate and citrate. Suitable antioxidants arethose as described herein, including bisulfite and sodium metabisulfite.Suitable local anesthetics include, but are not limited to, procainehydrochloride. Suitable suspending and dispersing agents are those asdescribed herein, including sodium carboxymethylcelluose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agentsinclude those described herein, including polyoxyethylene sorbitanmonolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamineoleate. Suitable sequestering or chelating agents include, but are notlimited to EDTA. Suitable pH adjusting agents include, but are notlimited to, sodium hydroxide, hydrochloric acid, citric acid, and lacticacid. Suitable complexing agents include, but are not limited to,cyclodextrins, including α-cyclodextrin, β-cyclodextrin,hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, andsulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, Kans.).

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms may be formulated for single or multiple dosageadministration. The single dosage formulations are packaged in anampule, a vial, or a syringe. The multiple dosage parenteralformulations must contain an antimicrobial agent at bacteriostatic orfungistatic concentrations. All parenteral formulations must be sterile,as known and practiced in the art.

In one embodiment, the pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated asready-to-use sterile solutions. In another embodiment, thepharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms are formulated as sterile dry soluble products, includinglyophilized powders and hypodermic tablets, to be reconstituted with avehicle prior to use. In yet another embodiment, the pharmaceuticalcomposition comprising a compound disclosed herein, a derivative oranalog thereof, including pharmaceutical salt forms and prodrug formsare formulated as ready-to-use sterile suspensions. In yet anotherembodiment, the pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated as steriledry insoluble products to be reconstituted with a vehicle prior to use.In still another embodiment, the pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms are formulated asready-to-use sterile emulsions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form” as used herein, refers to physically discrete unitssuited as unitary dosages for the individual to be treated; each unitcontaining a predetermined quantity of pharmaceutical composition iscalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The specification for the dosageunit forms of the disclosure are related to the characteristics of thepharmaceutical composition and the particular therapeutic effect to beachieve.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, suitable for injectable use include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the pharmaceutical composition comprising acompound disclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, should be sterile andshould be fluid to the extent that easy syringability exists. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size, in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be typical to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating apharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug form in the required amount in an appropriate solvent with oneor a combination of ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating a pharmaceutical composition comprising a compounddisclosed herein, a derivative or analog thereof, includingpharmaceutical salt forms and prodrug forms, into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, can be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The pharmaceutical compositioncomprising a compound disclosed herein, a derivative or analog thereof,including pharmaceutical salt forms and prodrug forms, and otheringredients can also be enclosed in a hard or soft-shell gelatincapsule, compressed into tablets, or incorporated directly into theindividual's diet. For oral therapeutic administration, thepharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms, can be incorporated with excipients and used in the formof ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations can, of course, bevaried and can conveniently be between about 5% to about 80% of theweight of the unit.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: a binder, such as gum gragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agent,such as corn starch, potato starch, alginic acid, and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin, or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above type, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac, sugar, or both.A syrup or elixir can contain the agent, sucrose as a sweetening agent,methyl and propylparabens as preservatives, a dye, and flavoring, suchas cherry or orange flavor. Of course, any material used in preparingany dosage unit form should be pharmaceutically pure and substantiallynon-toxic/biocompatible in the amounts employed.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms are disclosed herein, may be formulated as immediate ormodified release dosage forms, including delayed-, sustained-, pulsed-,controlled, targeted-, and programmed-release forms.

A pharmaceutical composition comprising a compound disclosed herein, aderivative or analog thereof, including pharmaceutical salt forms andprodrug forms are disclosed herein, may be formulated as a suspension,solid, semi-solid, or thixotropic liquid, for administration as animplanted depot. In one embodiment, the pharmaceutical compositioncomprising a compound disclosed herein, a derivative or analog thereof,including pharmaceutical salt forms and prodrug forms are dispersed in asolid inner matrix, which is surrounded by an outer polymeric membranethat is insoluble in body fluids but allows the active ingredient in thepharmaceutical compositions to diffuse through.

Suitable inner matrixes include polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers, such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol, and cross-linked partiallyhydrolyzed polyvinyl acetate.

Suitable outer polymeric membranes include polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer.

A therapeutically effective amount can be measured as the amountsufficient to decrease a subject's symptoms (e.g., dermatitis or rash bymeasuring the frequency of severity of skin sores). Typically, thesubject is treated with an amount of a therapeutic compositioncomprising a compound disclosed herein, a derivative or analog thereof,including pharmaceutical salt forms and prodrug forms, sufficient toreduce a symptom of a disease or disorder by at least 50%, 90% or 100%.Generally, the optimal dosage will depend upon the disorder and factorssuch as the weight of the subject, the type of bacteria, virus or fungalinfection, the weight, sex, and degree of symptoms. Nonetheless,suitable dosages can readily be determined by one skilled in the art.Typically, a suitable dosage is 0.5 to 40 mg/kg body weight, e.g., 1 to8 mg/kg body weight.

The compounds disclosed herein may also be combined or used incombination with other agents useful in the treatment, prevention, oramelioration of one or more symptoms of various syndromes, disorders,and/or diseases. Or, by way of example only, the therapeuticeffectiveness of one of the compounds described herein may be enhancedby administration of an adjuvant (i.e., by itself the adjuvant may onlyhave minimal therapeutic benefit, but in combination with anothertherapeutic agent, the overall therapeutic benefit to the patient isenhanced).

Such other agents, adjuvants, or drugs, may be administered, by a routeand in an amount commonly used therefor, simultaneously or sequentiallywith a compound as disclosed herein. When a compound as disclosed hereindisclosed herein is used contemporaneously with one or more other drugs,a pharmaceutical composition containing such other drugs in addition tothe compound disclosed herein may be utilized, but is not required.Accordingly, the pharmaceutical compositions disclosed herein includethose that also contain one or more other active ingredients ortherapeutic agents (e.g., an inhibitor of TNF, an antibiotic, and thelike), in addition to a compound disclosed herein. Suitable antibioticsinclude aminoglycosides (e.g., gentamicin), beta-lactams (e.g.,penicillins and cephalosporins), quinolones (e.g., ciprofloxacin), andnovobiocin. Generally, the antibiotic is administered in a bactericidal,antiviral and/or antifungal amount. Their effects can also be augmentedby co-administration with an inhibitor of flavohemoglobin, (Helmick etal., Imidazole antibiotics inhibit the nitric oxide dioxygenase functionof microbial flavohemoglobin. Antimicrob Agents Chemother, 2005,49(5):1837-43, and Sud et al., Action of antifungal imidazoles onStaphylococcus aureus, Antimicrob Agents Chemother, 1982, 22(3):470-4),increasing the efficacy of NO-based S. aureus killing by macrophages,and optionally triple combination therapies comprising one squalenesynthase inhibitor, one flavohemoglobin (nitric oxide dioxygenase)inhibitor such as an azole (miconazole, econazole, clotrimazole, andketoconazole) and one antibiotic as described above, may be applied to apatient in need of therapy. In a certain embodiment, a compounddisclosed herein can be combined with one or more antibiotics,including, but not limited to, amoxicillin, ampicillin, arsphenamine,azithromycin, aztreonam, azlocillin, bacitracin, carbenicillin,cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cefdinir,cefditorin, cefepime, cefixime, cefoperazone, cefotaxime, cefoxitin,cefpodoxime, cefprozil, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime, chloramphenicol, cilastin, ciprofloxacin,clarithromycin, clindamycin, clofazimine, cloxacillin, colistin,dalfopristan, demeclocycline, dicloxacillin, dirithromycin, doxycycline,erythromycin, enafloxacin, enviomycin, ertepenem, ethambutol,flucloxacillin, fosfomycin, furazolidone, gatifloxacin, geldanamycin,gentamicin, herbimicin, imipenem, linezolid, lomefloxacin, loracarbef,mafenide, moxifloxacin, meropenem, metronidazole, mezlocillin,minocycline, mupirozin, nafcillin, neomycin, netilmicin, nitrofurantoin,norfloxacin, oxytetracycline, penicillin, piperacillin, platensimycin,polymixin B, prochlorperazine, prontocil, quinupristine, rifabutin,roxithromycin, spectinomycin, sulfacetamide, sulfamethizole,sulfamethoxazole, teicoplanin, telithromycin, tetracycline,thioacetazone, thioridazine, ticarcillin, tobramycin, trimethoprim,troleandomycin, trovafloxacin, and vancomycin.

In a further embodiment, a compound provided herein can be combined withone or more sepsis treatments known in the art, including, but notlimited to, antibiotics, vasopressors, and corticosteroids.

In yet a further embodiment, a compound provided herein can be combinedwith one or more steroidal drugs known in the art, including, but notlimited to, aldosterone, beclometasone, betamethasone,deoxycorticosterone acetate, fludrocortisone acetate, hydrocortisone(cortisol), prednisolone, prednisone, methylprenisolone, dexamethasone,and triamcinolone.

In certain embodiments, a compound disclosed herein can be combined withone or more anti-fungal agents, including, but not limited to,amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine,butoconazole, caspofungin, ciclopirox, clotrimazole, econazole,fenticonazole, filipin, fluconazole, isoconazole, itraconazole,ketoconazole, micafungin, miconazole, naftifine, natamycin, nystatin,oxyconazole, ravuconazole, posaconazole, rimocidin, sertaconazole,sulconazole, terbinafine, terconazole, tioconazole, and voriconazole.

The compounds disclosed herein can also be administered in combination,preferably sequentially, with other classes of compounds, including, butnot limited to, antipruritics; anticoagulants, such as bivalirudin;thrombolytics, such as streptokinase; non-steroidal anti-inflammatoryagents, such as aspirin; antiplatelet agents, such as clopidogrel;norepinephrine reuptake inhibitors (NRIs) such as atomoxetine; dopaminereuptake inhibitors (DARIs), such as methylphenidate;serotonin-norepinephrine reuptake inhibitors (SNRIs), such asmilnacipran; sedatives, such as diazepham; norepinephrine-dopaminereuptake inhibitor (NDRIs), such as bupropion;serotonin-norepinephrine-dopamine-reuptake-inhibitors (SNDRIs), such asvenlafaxine; monoamine oxidase inhibitors, such as selegiline;hypothalamic phospholipids; endothelin converting enzyme (ECE)inhibitors, such as phosphoramidon; opioids, such as tramadol;thromboxane receptor antagonists, such as ifetroban; potassium channelopeners; thrombin inhibitors, such as hirudin; growth factor inhibitors,such as modulators of PDGF activity; platelet activating factor (PAF)antagonists; anti-platelet agents, such as GPIIb/IIIa blockers (e.g.,abdximab, eptifibatide, and tirofiban), P2Y(AC) antagonists (e.g.,clopidogrel, ticlopidine and CS-747), and aspirin; anti-coagulants, suchas warfarin; low molecular weight heparins, such as enoxaparin; FactorVIa Inhibitors and Factor Xa Inhibitors; renin inhibitors; neutralendopeptidase (NEP) inhibitors; vasopepsidase inhibitors (dual NEP-ACEinhibitors), such as omapatrilat and gemopatrilat; HMG CoA reductaseinhibitors, such as pravastatin, lovastatin, atorvastatin, simvastatin,NK-104 (a.k.a. itavastatin, nisvastatin, or nisbastatin), and ZD-4522(also known as rosuvastatin, or atavastatin or visastatin); squalenesynthetase inhibitors; fibrates; bile acid sequestrants, such asquestran; niacin; anti-atherosclerotic agents, such as ACAT inhibitors;MTP Inhibitors; calcium channel blockers, such as amlodipine besylate;potassium channel activators; alpha-adrenergic agents; diuretics, suchas chlorothlazide, hydrochiorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methylchlorothiazide,trichioromethiazide, polythiazide, benzothlazide, ethacrynic acid,tricrynafen, chlorthalidone, furosenilde, musolimine, bumetanide,triamterene, amiloride, and spironolactone; thrombolytic agents, such astissue plasminogen activator (tPA), recombinant tPA, streptokinase,urokinase, prourokinase, and anisoylated plasminogen streptokinaseactivator complex (APSAC); anti-diabetic agents, such as biguanides(e.g. metformin), glucosidase inhibitors (e.g., acarbose), insulins,meglitinides (e.g., repaglinide), sulfonylureas (e.g., glimepiride,glyburide, and glipizide), thiozolidinediones (e.g. troglitazone,rosiglitazone and pioglitazone), and PPAR-gamma agonists;mineralocorticoid receptor antagonists, such as spironolactone andeplerenone; growth hormone secretagogues; aP2 inhibitors;phosphodiesterase inhibitors, such as PDE III inhibitors (e.g.,cilostazol) and PDE V inhibitors (e.g., sildenafil, tadalafil,vardenafil); protein tyrosine kinase inhibitors; anti-inflammatories;anti-proliferatives, such as methotrexate, FK506 (tacrolimus, Prograf),mycophenolate mofetil; chemotherapeutic agents; immunosuppressants;anticancer agents and cytotoxic agents (e.g., alkylating agents, such asnitrogen mustards, alkyl sulfonates, nitrosoureas, ethylenimines, andtriazenes); anti-metabolites, such as folate antagonists, purineanalogues, and pyrridine analogues; antibiotics, such as anthracyclines,bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes, such asL-asparaginase; farnesyl-protein transferase inhibitors; hormonalagents, such as glucocorticoids (e.g., cortisone),estrogens/antiestrogens, androgens/antiandrogens, progestins, andluteinizing hormone-releasing hormone anatagonists, and octreotideacetate; microtubule-disruptor agents, such as ecteinascidins;microtubule-stablizing agents, such as pacitaxel, docetaxel, andepothilones A-F; plant-derived products, such as vinca alkaloids,epipodophyllotoxins, and taxanes; and topoisomerase inhibitors;prenyl-protein transferase inhibitors; and cyclosporins; cytotoxicdrugs, such as azathiprine and cyclophosphamide; TNF-alpha inhibitors,such as tenidap; anti-TNF antibodies or soluble TNF receptor, such asetanercept, rapamycin, and leflunimide; and cyclooxygenase-2 (COX-2)inhibitors, such as celecoxib and rofecoxib; and miscellaneous agentssuch as, hydroxyurea, procarbazine, mitotane, hexamethylmelamine, goldcompounds, platinum coordination complexes, such as cisplatin,satraplatin, and carboplatin.

The disclosure provides a method for inhibiting a bacterial, viral,parasitic and/or fungal-associated disorder by contacting oradministering a therapeutically effective amount of a compound disclosedherein, derivative or analog thereof either alone or in combination withother antimicrobial agents to a subject who has, or is at risk ofhaving, such a disorder. The term “inhibiting” means preventing orameliorating a sign or symptoms of a syndrome, disorder, and/or disease(e.g., a rash, sore, and the like). Examples of disease signs that canbe ameliorated include an increase in a subject's blood level of TNF,fever, hypotension, neutropenia, leukopenia, thrombocytopenia,disseminated intravascular coagulation, adult respiratory distresssyndrome, shock, rash, and organ failure. Examples of subjects who canbe treated in the disclosure include those at risk for, or thosesuffering from, a toxemia, such as endotoxemia resulting from agram-negative or gram-positive bacterial infection. Other examplesinclude subjects having dermatitis as well as those having skininfections or injuries subject to infection with gram-positive orgram-negative bacteria, a virus, or a fungus. Examples of candidatesubjects include those suffering from infection by E. coli, Neisseriameningitides, staphylococci, or pneumococci. Other subjects includethose suffering from gunshot wounds, renal or hepatic failure, trauma,burns, immunocompromising infections (e.g., HIV infections),hematopoietic neoplasias, multiple myeloma, Castleman's disease orcardiac myxoma. Those skilled in the art of medicine can readily employconventional criteria to identify appropriate subjects for treatment inaccordance with the disclosure.

The disclosure also provides a method for inhibiting the growth of abacterium by contacting the bacterium with compound, derivative oranalog thereof, including pharmaceutical salt and prodrug forms, with aninhibiting effective amount. The term “contacting” refers to exposingthe microbe (e.g., bacterium) to an agent so that the agent can inhibit,kill, or lyse microbe or render it susceptible to oxidative destruction.

Contacting can occur in vivo, for example, by administering thecompound, derivative or analog thereof, including pharmaceutical saltand prodrug forms, to a subject afflicted with a bacterial infection oris susceptible to a bacterial infection. In vivo contacting includesboth parenteral as well as topical. “Inhibiting” or “inhibitingeffective amount” refers to the amount of agent that is sufficient tocause, for example, a bacteriostatic or bactericidal effect. Bacteriathat can be affected by the use of a compound, derivative or analogthereof, including pharmaceutical salt and prodrug forms, include bothgram-negative and gram-positive bacteria. For example, bacteria that canbe affected include Staphylococcus aureus, Streptococcus pyogenes (groupA), Streptococcus sp. (viridans group), Streptococcus agalactiae (groupB), S. bovis, Streptococcus (anaerobic species), Streptococcuspneumoniae, and Enterococcus sp.; Gram-negative cocci such as, forexample, Neisseria gonorrhoeae, Neisseria meningitidis, and Branhamellacatarrhalis; Gram-positive bacilli such as Bacillus anthracis, Bacillussubtilis, P. acne Corynebacterium diphtheriae and Corynebacteriumspecies which are diptheroids (aerobic and anerobic), Listeriamonocytogenes, Clostridium tetani, Clostridium difficile, Escherichiacoli, Enterobacter species, Proteus mirablis and other sp., Pseudomonasaeruginosa, Klebsiella pneumoniae, Salmonella, Shigella, Serratia, andCampylobacter jejuni. Infection with one or more of these bacteria canresult in diseases such as bacteremia, pneumonia, meningitis,osteomyelitis, endocarditis, sinusitis, arthritis, urinary tractinfections, tetanus, gangrene, colitis, acute gastroenteritis, impetigo,acne, acne posacue, wound infections, born infections, fascitis,bronchitis, and a variety of abscesses, nosocomial infections, andopportunistic infections. The method for inhibiting the growth ofbacteria can also include contacting the bacterium with a compounddisclosed herein, derivative or analog, including pharmaceutical saltand prodrug forms, thereof in combination with one or more antibiotics.

Fungal organisms may also be affected by a compound disclosed herein,derivative or analog thereof, including pharmaceutical salt and prodrugforms, include for example, Microsporum canis and other Microsporum sp.;Trichophyton sp. such as T. rubrum, and T. mentagrophytes, yeasts, e.g.,Candida albicans, C. Tropicalis, or other Candida species, Saccharomycescerevisiae, Torulopsis glabrata, Epidermophyton floccosum, Malasseziafurfur (Pityropsporon orbiculare, or P. ovale, Cryptococcus neoformans,Aspergillus fumigatus, Aspergillus nidulans, and other Aspergillus sp.,Zygomycetes, e.g., Rhizopus, Mucor, Paracoccidioides brasiliensis,Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis,and Sporothrix schenckii.

The methods and compositions of the disclosure utilizing Firmocidincompound, derivative or analog thereof are useful as a broad-spectrumantimicrobials suitable for tackling the growing problem ofantibiotic-resistant bacteria strains, and for treating and/orpreventing outbreaks of infectious diseases, including diseases causedby bioterrorism agents like anthrax, plague, cholera, gastroenteritis,multidrug-resistant tuberculosis (MDR TB).

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. Such kits cancomprise a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic.

For example, the container(s) can comprise one or more compoundsdescribed herein, optionally in a composition or in combination withanother agent as disclosed herein. The container(s) optionally have asterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). Such kits optionally comprise a compound disclosedherein with an identifying description or label or instructions relatingto its use in the methods described herein.

A kit will typically comprise one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but are not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

A label can be on or associated with the container. A label can be on acontainer when letters, numbers or other characters forming the labelare attached, molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Alabel can be used to indicate that the contents are to be used for aspecific therapeutic application. The label can also indicate directionsfor use of the contents, such as in the methods described herein. Theseother therapeutic agents may be used, for example, in the amountsindicated in the Physicians' Desk Reference (PDR) or as otherwisedetermined by one of ordinary skill in the art. The invention is furtherillustrated by the following examples:

EXAMPLES

Initial purification involved 5 steps including three modes ofchromatography. High-resolution ES-mass spectrometry analysis predictedthe antibiotic component to have a molecular formula of C₅H₅N₅O. Thechemical shifts obtained by NMR structural analyses were found tocorrelate to the shifts of adenine-N oxide. Based on the structuralanalyses, the compound, firmocidin, did not match any prior knownantimicrobial agents. When S. epidermidis was cultured in the presenceof ammonium-¹⁵N chloride, the isotope was incorporated into firmocidin,indicating that firmocidin is produced via de novo synthesis and by thefermentation of culture media. Firmocidin killed 99.75% of group Astreptococcus, 99.12% of group B streptococcus and 96.84% ofStaphylococcus aureus when used at 60 μg/ml for 3 h. Firmocidin alsosuppressed growth of methicillin-resistant S. aureus strains. Itsantimicrobial activity was stronger in these assays than theantimicrobial, benzoyl peroxide, which is one of the most frequentlyused active ingredients in topically administered therapeutics. However,firmocidin did not affect the growth of S. epidermidis itself.Firmocidin also did not affect viability of human keratinocytes andsebocytes in culture. Thus, firmocidin shows pathogen-specific activityand low toxicity while maintaining S. epidermidis viability andpotentially a normal microbiome. Our data support the hypothesis that S.epidermidis contributes to skin innate immune defense and suggests anovel therapeutic approach to normalize skin bacterial colonization.

Purification of Firmocidin.

Several S. epidermidis strains were clinically isolated from healthyhuman skin and screened by radial diffusion antimicrobial assays.Extracts form S. epidermidis strain MO34 were found to have the highestantimicrobial activity. Consequently, S. epidermidis strain MO34 wasused for all future studies. S. epidermidis strain MO34 bacteria werecultured in reduced media comprised of 25% tryptic soy broth (Sigma) at37° C. for 24 hours. The supernatant was then purified from thebacterial cells by filtering the bacterial culture through a 0.2 μmfilter. After all liquid from the supernatant was removed in vacuo, theresulting residue was re-suspended in methanol. The supernatant wascentrifuged, dried, and then partitioned by adding 90% acetonitrile/10%water. The organic layer was collected and dried under nitrogen gas. Theresulting residue was dissolved in 2% acetonitrile/98% water and thenloaded onto a C₁₈ sep-pak cartridge (10 g; Waters Inc.). The cartridgewas washed with 2% acetonitrile (35 mL), followed by elution with 2%acetonitrile (40 mL). The eluent was dried, and then re-suspended in100% acetonitrile. The resulting crude product was then purified byusing HPLC with a Taskgel NH₂-100 column (4.5 mm×150 mm; TosohBioscience) in hydrophilic-interaction (HILIC) mode. The title productwas eluted by using a linear gradient of 5-20% water in acetonitrile ata flow rate of 1 mL/min; the eluent was monitored at 270 nm. Fractions26-31 were lyophilized, reconstituted in PBS and applied on an agarplate inoculated with GAS (NZ131). Fractions purified from the S.epidermidis strain were screened by radial diffusion antimicrobialassays. Clear zones on the agar plates indicate antimicrobial activityof the fractions. It was found that fraction #28, which correlated to apeak on the chromatogram, showed significant antimicrobial activity (seee.g., FIG. 2). When S. epidermidis straine MO34 was cultured in thepresence of ammonium-¹⁵N chloride, the isotope was incorporated intofirmocidin, indicating that firmocidin is produced via de novo synthesisand not by fermentation of culture media. Approximately, 7 mg of theantibiotic component from fraction #28 was purified from culturesupernatant (6 L) for use in future studies.

Mass Spectroscopy.

High-resolution ES-MS analysis gave the molecular formula C₅H₅N₅O(calculated exact mass for [M+H]⁺=152.0567, observed mass=152.0567) (seee.g., FIG. 3-4). In addition, ESI positive ion mode MS/MS analysis onthe peak of 152.0567 predicts that firmocidin has at least one hydroxylgroup (see e.g., FIG. 4B). The fragmentation profile of firmocidin didnot match 6-hydroxyamino purine or guanine in electron-impact massspectrometry experiments (see e.g., FIGS. 5-8). While data produced frommass spectrometry are typically reproducible and consistent, the dataproduced, such as the m/z ion fragment peaks, can be affected by samplepreparation, temperature, quality of calibration, differences in devicesutilized, etc. Accordingly, the data presented in FIGS. 3-8 canpotentially vary up to 10 milli mass units.

¹H and ¹²C NMR Spectrum.

1H NMR spectra were recorded at 20° C. at 500 MHz with a Bruker Avance750 NMR instrument spectrometer. HPLC-purified firmocidin was dissolvedin deuterium oxide (100%) (Cambridge isotope Inc.) at 2 mg/mL. The ¹Hspectrum of firmocidin has nuclear shifts at 8.0-8.5 ppm suggesting thatfirmocidin has a benzene ring (see e.g., FIG. 10). In addition, thechemical shifts from the 2D-NMR studies indicate that firmocidin is anovel antimicrobial agent, in that the chemical shifts did not match anypreviously characterized antimicrobial agent (see e.g., FIGS. 9-10). Thechemical shifts obtained by NMR structural analyses, however, correlatedto the shifts seen with an adenine-N oxide. While data produced from NMRare generally reproducible and consistent, the data produced can beaffected by sample preparation, temperature, differences in devicesutilized, etc. Accordingly, the data generated, such as the ppm peaks,presented in FIGS. 9-10 can vary by plus or minus one ppm.

In Vitro Antibacterial Studies.

Purified firmocidin was prepared at minimal bactericidal concentrations.The in-vitro time-bacteria kill curves were then determined in thepresence of carbonate. But since Group A Streptococcus (GAS) will notgrow in media containing carbonate (Dorschner et al., 2006), thein-vitro time-GAS kill curves were determined in media (25% Todd-HewittBroth, 75% of 1× Dulbecco's phosphate-buffered saline) that did notcontain carbonate. As a control for the GAS in-vitro time-bacteria killcurve, GAS and S. epidermidis were grown in the same medium.

Purified firmocidin suppresses growth of GAS, group B streptococcus(GBS), methicillin-sensitive S. aureus and MRSA, but not S. epidermidis(see e.g., FIG. 12) and Gram negative bacteria. Its antimicrobialactivity was stronger than benzoyl peroxide (BPO), which is an activeingredient in topically applied medications to treat various conditions,including e.g. acne vulgaris (see e.g., FIG. 12). Furthermore, thein-vitro time-bacteria kill curves demonstrate that firmocidin exhibitsbactericidal activity against GAS and bacteriostatic activity againstGBS and S. aureus (see FIG. 13), but not against S. epidermidis.Firmocidin killed 99.75% of group A streptococcus, 99.12% of group Bstreptococcus and 96.84% of Staphylococcus aureus when used at 60 μg/mlfor 3 h. Firmocidin also suppressed growth of methicillin-resistant S.aureus strains. It would be expected that firmocidin would have similarantimicrobial activity against other pathogens which have not beentested. Such pathogens, including but not limited to, Streptococcus sp.(viridans group), S. bovis, Streptococcus (anaerobic species),Streptococcus pneumoniae, and Enterococcus sp.; Gram-negative cocci suchas, for example, Neisseria gonorrhoeae, Neisseria meningitidis, andBranhamella catarrhalis; Gram-positive bacilli such as Bacillusanthracis, Bacillus subtilis, P. acne Corynebacterium diphtheriae andCorynebacterium species which are diptheroids (aerobic and anerobic),Listeria monocytogenes, Clostridium tetani, Clostridium difficile,Escherichia coli, Enterobacter species, Proteus mirablis and other sp.,Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, Shigella,Serratia, Campylobacter jejuni, Microsporum canis and other Microsporumsp.; Trichophyton sp. such as T. rubrum, and T. mentagrophytes, yeasts,e.g., Candida albicans, C. Tropicalis, or other Candida species,Saccharomyces cerevisiae, Torulopsis glabrata, Epidermophyton floccosum,Malassezia furfur (Pityropsporon orbiculare, or P. ovale, Cryptococcusneoformans, Aspergillus fumigatus, Aspergillus nidulans, and otherAspergillus sp., Zygomycetes, e.g., Rhizopus, Mucor, Paracoccidioidesbrasiliensis, Blastomyces dermatitides, Histoplasma capsulatum,Coccidioides immitis, and Sporothrix schenckii, can be tested by usingthe methods provided in this disclosure, or by making obvious variationsthereof, including using pathogen specific growth media, pathogenspecific growth conditions, etc. Such experiments would be consideredroutine to one in the skill in the art, in that they are commonlyperformed and there is extensive documentation with respect to theproper growth conditions for specific pathogenic organisms.

Cytotoxic Effect of Firmocidin.

The immortalized human HaCaT keratinocytes and SZ95 sebocytes were grownto 75% confluence (1×10⁵ cell/well) and were incubated with variousconcentrations of firmocidin for 24 hours. As a background, Triton X-100(0.1% (v/v)) was added to achieve 0% cell viability. After incubation,cell viabilities were determined with a cell viability assay kit(Promega) according to the instruction provided.

The cell viability studies indicate that firmocidin is not cytotoxic tohuman keratinocytes and sebocytes (see FIG. 14). The data suggests thatfirmocidin is safe to use on subjects. Thus, firmocidin ispathogen-specific, and a safe antimicrobial therapy for infectionscaused by GAS, GBS and S. aureus. Furthermore, additional routineexperiments may be performed using the methods provided herein, toperform in vitro cell viability studies with other cell lines.

In Vivo Studies in a Murine Model.

ED50 evaluations are carried out in CFl female mice injectedintraperitoneally with sufficient bacteria to kill 100% of the untreatedanimals for all methicillin-sensitive and methicillin-resistant S.aureus strains. C₃H/HeN female mice are utilized in the tests for GASand GBS studies. Thawed bacterial cultures are suspended in BHI brothwhich contained 4-8% dried Brewer's yeast (w/v). The infecting inoculum(0.2 mL) is adjusted to yield ca. 100 times the 50% lethal dose (LD50).Concurrently with each trial, the challenge LD50 is validated byinoculating untreated animals with log dilutions of the bacteria. Fivedosage levels representing a 5 log dilution range are employed perdetermination with 10 mice utilized at each level. A mortality rate of90-100% is produced in all groups of untreated mice with the 100×LD50challenge inoculum. Test compounds are formulated in water or saline,with gentle heating at higher concentrations, and administered orally orsubcutaneously at 1 and 5 hours post-infection. At least five dosagelevels of firmocidin utilizing serial 2-fold dilutions are employed foreach ED50 determination. One treatment group of six mice is used foreach firmocidin dosage level. Deaths in each group following infectionand treatment are monitored daily for at least 6 days. Following thisobservation period, cumulative mortality figures are used to calculateby probit analysis the amount of drug in mg of drug/kg of body

Antimicrobial peptides serve as a first line of innate immune defenseagainst invading organisms such as bacteria and viruses. The disclosuredemonstrates that antibiotics are produced by a normal microbialresident of human skin, Staphylococcus epidermidis, which act as anantimicrobial shield and contribute to normal defense at the epidermalinterface. An agent was obtained with antimicrobial activity fromculture supernatant of S. epidermidis MO34, a strain isolated fromhealthy human skin surface. According to the NMR structure analysis, thecomponent was a novel antibiotic of which chemical formula was C₅H₅N₅Oreferred to herein as firmocidin. Firmocidin exhibited bactericidalactivity against group A streptococcus and bacteriostatic activityagainst group B streptococcus and Staphylococcus aureus. Most notably,firmocidin suppresses growth of methicillin-resistant S. aureus (MRSA),a strain that is highly resistant to some antibiotics. However,firmocidin does not affect the growth of S. epidermidis whichcontributes normal defense at the skin epithelium. Firmocidin also didnot adversely affect the viability of human keratinocytes and sebocytes.In addition, firmocidin is isolated from a microorganism residing in thenormal skin microflora, suggesting low toxicity to the host. Thus,firmocidin may have potential to be safely used as a pathogen-specificantibiotic therapy for skin infections.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-14. (canceled)
 15. A composition comprising a supernatant of culturedStaphylococcus epidermidis strain MO34 purified from bacterial cells byfiltering a culture of Staphylococcus epidermidis strain MO34 though a0.2 um filter.
 16. The composition of claim 15, wherein said supernatantis dried under vacuum.
 17. The composition of claim 15, wherein saidcomposition is formulated for topical administration.
 18. Thecomposition of claim 16, wherein said supernatant is incorporated into aliposome, micelle, microsphere, nanosystem, or a mixture thereof. 19.The composition of claim 16, wherein said supernatant is incorporatedinto a lotion, cream, soap, or wipe.
 20. The composition of claim 16,wherein said supernatant is incorporated into an emulsion, solution,suspension, gel, hydrogel, ointment, dusting powder, dressing, elixir,tincture, paste, foam, film, aerosol, irrigation, spray, suppository,bandage, or dermal patch.
 21. The composition of claim 15, wherein saidculture of Staphylococcus epidermidis strain MO34 is grown in trypticsoy broth.
 22. A method of making an antimicrobial compositioncomprising: (a) culturing Staphylococcus epidermidis strain MO34 in aculture medium; (b) isolating the culture supernatant; and (c)lyophilizing said supernatant.
 23. The method of claim 22, wherein theculture supernatant is isolated by filtration.
 24. The method of claim23, wherein said filtration utilizes a 0.2 um filter.
 25. The method ofclaim 23, wherein said filtration utilizes a filter with a 3 kilodaltonmolecular weight cutoff.
 26. A method of inhibiting a skin pathogencomprising contacting said skin pathogen with the composition of claim15.
 27. The method of claim 26, wherein said skin pathogen is selectedfrom the group consisting of: Staphylococcus aureus, Streptococcuspyogenes (group A), Streptococcus sp. (viridans group), Streptococcusagalactiae (group B), S. bovis, Streptococcus (anaerobic species),Streptococcus pneumoniae, Enterococcus sp., Gram-negative cocci,Neisseria gonorrhoeae, Neisseria meningitidis, Branhamella catarrhalis,Gram-positive bacilli such as Bacillus anthracis, Bacillus subtilis, P.acnes Corynebacterium diphtheria, Corynebacterium species which arediptheroids (aerobic and anerobic), Listeria monocytogenes, Clostridiumtetani, Clostridium difficile, Escherichia coli, Enterobacter species,Proteus mirablis, Proteus sp., Pseudomonas aeruginosa, Klebsiellapneumoniae, Salmonella, Shigella, Serratia, Campylobacter jejuni, GroupA streptococcus (GAS), Group B streptococcus (GBS), S. aureus,Microsporum sp., Trichophyton sp., T. rubrum, T. mentagrophytes, yeasts,Candida albicans, C. Tropicalis, Candida sp., Saccharomyces cerevisiae,Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur,Pityropsporon orbiculare, P. ovale, Cryptococcus neoformans, Aspergillusfumigatus, Aspergillus nidulans, Aspergillus sp., Zygomycetes, Rhizopussp., Mucor sp, Paracoccidioides brasiliensis, Blastomyces dermatitides,Histoplasma capsulatum, Coccidioides immitis, and Sporothrix schenckiiand any combinations thereof.
 28. The method of claim 26, wherein saidskin pathogen is contacted with the composition of claim
 16. 29. Themethod of claim 26, wherein said skin pathogen is contacted with thecomposition of claim
 17. 30. The method of claim 26, wherein said skinpathogen is contacted with the composition of claim
 18. 31. The methodof claim 26, wherein said skin pathogen is contacted with thecomposition of claim
 19. 32. The method of claim 26, wherein said skinpathogen is contacted with the composition of claim
 20. 33. The methodof claim 26, wherein said skin pathogen is contacted with thecomposition of claim 21.